US009856497B2 (12 ) United States Patent ( 10 ) Patent No. : US 9 ,856 ,497 B2 Qi et al. (45 ) Date of Patent: Jan . 2 , 2018

(54 ) CHIMERIC AND METHODS OF 9 , 593 , 338 B2 3 / 2017 Liu et al . REGULATING EXPRESSION 9 ,624 ,554 B2 4 /2017 Collins et al . 2004/ 0197346 Al 10 / 2004 O 'Hare et al. 2006 /0182741 A1 8 / 2006 Bourel et al. @( 71 ) Applicant: THE BOARD OF TRUSTEES OF 2007 /0224615 Al 9 /2007 Lee et al. THE LELAND STANFORD JUNIOR 2007 /0231319 A1 10 /2007 Yednock et al. UNIVERSITY , Stanford , CA (US ) 2008 /0274913 AL 11 / 2008 Lee 2010 / 0076057 A1 3 / 2010 Sontheimer et al. 2010 / 0093617 AL 4 / 2010 Barrangou et al. @( 72 ) Inventors : Lei S . Qi, Stanford , CA (US ) ; P . C . 2011 / 0217739 AL 9 / 2011 Terns et al. Dave P . Dingal, Stanford , CA (US ) 2012 / 0077706 A1 * 3 / 2012 Lee ...... C12N 15 / 1055 506 / 11 @( 73 ) Assignee: The Board of Trustee of the Leland 2013 /0011828 A1 1 / 2013 Barrangou et al. Stanford Junior University , Stanford , 2014 / 0068797 Al 3 / 2014 Doudna et al. CA (US ) 2014 /0113376 Al 4 /2014 Sorek et al. 2014 /0179770 A1 * 6 /2014 * ZhangLilang ...... C12N 15 / 86 @ 514 /44 R ( * ) Notice: Subject to any disclaimer, the term of this 2014 /0199767 Al 7 / 2014 Barrangou et al . patent is extended or adjusted under 35 2014 /0234851 A1 8 / 2014 Leonard et al. U . S .C . 154 (b ) by 0 days . 2014 /0271635 Al 9 /2014 Brogdon et al . 2014 /0273226 AL 9 /2014 Wu ( 21) Appl. No .: 15 /403 , 058 2014 / 0294773 A1 10 / 2014 Brouns et al. 2014 /0295556 Al 10 /2014 Joung et al. 2014 / 0295557 A1 10 /2014 Joung et al . (22 ) Filed : Jan . 10 , 2017 2014 / 0315985 A 10 / 2014 May et al . 2014 /0342456 AL 11 /2014 Mali et al . (65 ) Prior Publication Data 2014 /0342457 AL 11/ 2014 Mali et al . 2014 /0342458 A1 11 /2014 Mali et al . US 2017 /0198308 A1 Jul. 13, 2017 (Continued ) Related U .S . Application Data FOREIGN PATENT DOCUMENTS ( 60 ) Provisional application No . 62/ 277 , 322 , filed on Jan . 11 , 2016 , provisional application No . 62 /351 ,522 , CA 2619833 Al 3 / 2007 filed on Jun . 17 , 2016 , provisional application No . EP 1916903 B1 7 /2010 62 /399 ,902 , filed on Sep . 26 , 2016 . (Continued ) (51 ) Int. CI. OTHER PUBLICATIONS CO7H 21/ 04 (2006 .01 ) ( 2006 .01 ) Cheng et al ( Research , 2013 , 23 : 1163 - 1171 ). * C12N 15 /90 Chavez et al (Nature Methods, 2015 , 12 : 326 - 310 ). * C12N 9 / 22 ( 2006 .01 ) Nunez et al ( ACS Chem . Biol .; Feb . 9 , 2016 ; 11: 681 -688 ). * ( 52 ) U .S . CI. Invitrogen “ TangoTM GPR21 - bla U2OS Cell -based Assay , " printed CPC ...... C12N 15 / 907 (2013 .01 ) ; C12N 9 /22 2010 , p . 1 - 12 . ( 2013 .01 ) ; C12Y 301/ 00 (2013 .01 ) Lim et al (Nature Reviews, 2010 , 11: 393 -403 ) * ( 58 ) Field of Classification Search Daringer et al (ACS Synthetic , 2014 , 3 :892 - 902 ). * None Wehr et al ( Chapter 8 ; G - Coupled Screening See application file for complete search history . Assays, Methods in Molecular Biology , Jan . 2015 ; vol. 1272 , p . 107 - 118 ). * (56 ) References Cited Qi et al ( Cell, 2013, 152 : 1173 - 1183 ) .* U . S . PATENT DOCUMENTS (Continued ) 5 , 888 , 813 A 3 / 1999 Endl et al. 7 , 049 ,076 B2 . 5 / 2006 Lee et al . Primary Examiner — Laura B Goddard 8 , 017 ,398 B2 9 /2011 Lee et al. 8 , 546 , 553 B2 10 / 2013 Terns et al . (74 ) Attorney , Agent, or Firm — Kilpatrick Townsend & 8 , 697, 359 B1 4 / 2014 Zhang Stockton LLP 8 ,771 , 945 B1 . 7 / 2014 Zhang 8 , 795 , 965 B2 8 / 2014 Zhang 8 , 865 ,406 B2 10 / 2014 Zhang et al. (57 ) ABSTRACT 8 ,871 ,445 B2 10 / 2014 Le Cong et al . 8 , 889 , 356 B2 11/ 2014 Zhang The present disclosure provides systems, compositions and 8 , 895 ,308 B1 11/ 2014 Zhang et al. 8 , 906 , 616 B2 12 / 2014 Zhang et al. methods for regulating expression of a target polynucleotide 8 , 932 , 814 B2 1 /2015 Le Cong et al. in a cell. The systems, compositions and methods comprise 8, 945, 839 B2 2 /2015 Zhang a chimeric receptor polypeptide comprising a G -protein 8 ,993 ,233 B2 3 / 2015 Zhang et al. 8 , 999 , 641 B2 4 / 2015 Zhang et al. coupled receptor (GPCR ) or a fragment thereof, a chimeric 9 , 023 , 649 B2 5 / 2015 Mali et al . adaptor polypeptide, at least one actuator moiety and a 9 , 234 ,213 B2 1 / 2016 Wu cleavage moiety . 9 , 260 , 752 B1 2 / 2016 May et al. 9 , 580, 480 B2 2 / 2017 Lu et al. 9 , 587, 020 B2 3 /2017 Wu et al . 30 Claims, 49 Drawing Sheets US 9, 856 ,497 B2 Page 2

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( 56 ) References Cited OTHER PUBLICATIONS International Search Report dated May 15 , 2017 for PCT/ US2017 / 012881 filed Jan . 10 , 2017 ( 5 pgs . ) . International Search Report dated Jun . 29 , 2017 for corresponding PCT Appln No. PCT/ US2017 /012885 filed Jan . 10 , 2017 ( 6 pgs. ) . * cited by examiner U . S . Patent Jan . 2 , 2018 Sheet 1 of 49 US 9 ,856 ,497 B2

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onde buonnon O OH LI DAPT DAPT US 9 , 856 , 497 B2 CHIMERIC PROTEINS AND METHODS OF cleavage recognition site ; wherein : (i ) the GMP forms a REGULATING GENE EXPRESSION portion of an intracellular region of the chimeric receptor polypeptide, and the cleavage moiety forms a portion of the CROSS -REFERENCE chimeric adaptor polypeptide ; ( ii ) the GMP forms a portion 5 of the chimeric adaptor polypeptide , and the cleavage moi This application claims the benefit of U .S . Provisional ety forms a portion of an intracellular region of the chimeric Application No . 62 /277 . 322 filed on Jan . 11. 2016 . US. receptor polypeptide ; or ( iii ) the cleavage moiety is com Provisional Application No . 62 /351 , 522 filed on Jun . 17 . plexed with a second adaptor polypeptide that binds the 2016 , and U . S . Provisional Application No. 62 /399 . 902 filed chimeric receptor polypeptide in response to the receptor on Sep . 26 . 2016 . each of which is incorporated in its 10 modification , and the GMP forms a portion of the chimeric entirety herein by reference . adaptor polypeptide . In some embodiments , the receptor does not comprise SEQ ID NO : 39 . REFERENCE TO A SEQUENCE LISTING In some embodiments , the target polynucleotide is genomic DNA . In some embodiments , the target polynucle The Sequence Listing written in file 079445 - 000830US- 15 otide is RNA . In some embodiments , the modification is 1015078 SequenceListing . txt, created on Mar. 8 , 2017 , 189 , . 094 bytes, machine format IBM -PC , MS - Windows operat In some embodiments , the actuator moiety is a Cas ing system , is hereby incorporated by reference in its protein , and the system further comprises a guide RNA entirety for all purposes active to form a complex with the Cas protein . In some 20 embodiments , ( i ) the actuator moiety is an RNA binding BACKGROUND protein (RBP ) optionally complexed with a guide RNA , and ( ii ) the system further comprises a Cas protein that is able to Regulation of cell activities can involve the binding of a form a complex with the guide RNA . In some embodiments , ligand to a membrane -bound receptor comprising an extra - the Cas protein substantially lacks DNA cleavage activity . cellular ligand binding domain and an intracellular ( e . g ., 25 In some embodiments , ( i ) the GMP forms a portion of the cytoplasmic ) signaling domain . The formation of a complex chimeric adaptor polypeptide , ( ii ) cleavage of the cleavage between a ligand and the ligand binding domain can result recognition site is effective to release the chimeric adaptor in a conformational and /or chemical modification in the polypeptide from the receptor, and ( iii ) the system comprises receptor which can result in a signal transduced within the a further chimeric adaptor polypeptide comprising an GMP cell. In some situations, the cytoplasmic portion of the 30 that binds to the modified receptor. receptor is phosphorylated ( e . g ., trans - and /or auto -phos - In some embodiments , receptor modification comprises phorylated ) , resulting in a change in its activity . These modification at multiple modification sites, and each modi events can be coupled with secondary messengers and / or the fication site is effective to bind an adaptor polypeptide . recruitment of co - factor proteins. In some instances , the In some embodiments , the cleavage recognition site com change in the cytoplasmic portion results in binding to other 35 prises a polypeptide sequence , and the cleavage moiety proteins ( e . g . , co - factor proteins and / or other receptors ) . comprises protease activity . In some embodiments , the These other proteins can be activated and then carry out cleavage recognition site comprises a disulfide bond , and the various functions within a cell . cleavage moiety comprises oxidoreductase activity . In some Conditional gene expression systems allow for condi embodiments , the cleavage recognition site comprises a first tional regulation of one or more target genes . Conditional 40 portion of an intein sequence that reacts with a second gene expression systems such as drug - inducible gene portion of the intein sequence to release the actuator moiety . expression systems allow for the activation and /or deacti In some embodiments , the receptor is a transmembrane vation of gene expression in response to a stimulus , such as receptor. In some embodiments , the receptor is a nuclear the presence of a drug . Currently available systems, how - receptor. ever, can be limited due to imprecise control, insufficient 45 In some embodiments , the actuator moiety regulates levels of induction ( e . g . , activation and /or deactivation of expression of the target polynucleotide by physical obstruc gene expression ), and lack of specificity . tion of the target polynucleotide or recruitment of additional factors effective to suppress or enhance expression of the SUMMARY target polynucleotide . In some embodiments, the actuator 50 moiety comprises an activator effective to increase expres In view of the foregoing , there exists a considerable need sion of the target polynucleotide . In some embodiments , the for alternative compositions and methods to carry out con - actuator moiety is linked to at least one nuclear localization ditional regulation of gene expression , for example by signal (NLS ) . regulating expression of a target polynucleotide . In an In some embodiments , the chimeric receptor polypeptide aspect, the present disclosure provides a system for regu - 55 is linked to at least one targeting sequence which directs lating expression of a target polynucleotide in a cell. In some transport of the receptor to a specific region of a cell . In embodiments , the system comprises ( a ) a chimeric receptor some embodiments , the targeting sequence directs transport polypeptide that is modified upon binding an antigen , of the receptor to a nucleus, cytoplasm , mitochondria , endo wherein receptor modification comprises a conformational plasmic reticulum (ER ) , chloroplast, apoplast , peroxisome change or chemical modification ; ( b ) a chimeric adaptor 60 or plasma membrane . In some embodiments , the targeting polypeptide that binds the receptor in response to the recep - sequence comprises a nuclear export signal (NES ) . In some tor modification ; ( c ) a gene modulating polypeptide (GMP ) embodiments , the targeting sequence comprises a plasma comprising an actuator moiety linked to a cleavage recog- membrane targeting . nition site , wherein upon cleavage of the cleavage recogni - In some embodiments , the chimeric adaptor polypeptide tion site, the actuator moiety is activated to complex with a 65 is linked to at least one targeting sequence which directs target polynucleotide ; and ( d ) a cleavage moiety that cleaves transport of the adaptor to a specific region of a cell . In some the cleavage recognition site when in proximity to the embodiments , the targeting sequence directs transport of the US 9 ,856 , 497 B2 chimeric adaptor polypeptide to a nucleus, cytoplasm , mito In some embodiments , the receptor is a transmembrane chondria , (ER ) , chloroplast, apo receptor. In some embodiments, the receptor is a nuclear plast , peroxisome or plasma membrane . In some embodi - receptor. ments , the targeting sequence comprises a nuclear export In some embodiments , the actuator moiety regulates signal (NES ) . In some embodiments , the targeting sequence 5 expression of the target polynucleotide by physical obstruc comprises a plasma membrane targeting peptide . tion of the target polynucleotide or recruitment of additional In some embodiments , the receptor is linked to a poly factors effective to suppress or enhance expression of the target polynucleotide . In some embodiments , the actuator peptide folding domain . In some embodiments , the chimeric moiety comprises an activator effective to increase expres adaptor polypeptide is linked to a polypeptide folding 10 sion of the target polynucleotide . domain . In an aspect , the present disclosure provides a chimeric In an aspect , the present disclosure provides a method of . In some embodiments , the receptor regulating expression of a target polynucleotide in a cell . In comprises (a ) an antigen interacting domain that specifically some embodiments , the method comprises (a ) exposing a binds an antigen ; and (b ) an actuator moiety linked to the chimeric receptor polypeptide to an antigen , wherein ((i 1 ) thethe 1515 antigenanti interacting domain ; wherein : ( i) the chimeric intra receptor is modified upon exposure to the antigen , and ( ii ) cellular receptor is modified in response to antigen binding ; receptormodification comprises a conformational change or (ii ) the chimeric receptor polypeptide translocates to a a chemical modification ; ( b ) binding a chimeric adaptor nucleus of a cell in response to modification ; and ( iii ) the polypeptide to the chimeric receptor polypeptide in response actuator moiety complexes with a target polynucleotide in to receptor modification to form a complex between a gene 20 the nucleus . modulating polypeptide (GMP ) and a cleavage moiety , In some embodiments , the actuator moiety is a Cas wherein the GMP comprises an actuator moiety linked to a protein that forms a complex with a guide RNA . In some cleavage recognition site ; and (c ) cleaving the cleavage embodiments , the Cas protein substantially lacks DNA recognition site with the cleavage moiety , wherein upon cleavage activity . cleavage of the cleavage recognition site , the actuator moi - 25 In some embodiments , the actuator moiety regulates ety complexes with a target polynucleotide thereby regulat - expression of the target polynucleotide by physical obstruc ing expression of the target polynucleotide in the cell ; tion of the target polynucleotide or recruitment of additional factors effective to suppress or enhance expression of the wherein : (i ) the GMP forms a portion of an intracellular target polynucleotide . In some embodiments, the actuator region of the chimeric receptor polypeptide, and the cleaven 30 moiety comprises an activator effective to increase expres age moiety forms portion of the chimeric adaptor polypep - 30 sion of the target polynucleotide. tide ; (ii ) the cleavage moiety forms part of the chimeric In some embodiments , the antigen is a . adaptor polypeptide , and the GMP forms a portion of an In some embodiments , the actuator moiety is linked to at intracellular region of the chimeric receptor, or (iii ) the least one nuclear localization signal (NLS ) . cleavage moiety is complexed with a second adaptor poly - 35 In some embodiments , the receptor is linked to at least peptide that binds the receptor in response to the receptor one targeting sequence which directs transport of the recep modification , and the GMP forms a portion of the chimeric tor to a specific region of a cell. In some embodiments, the adaptor polypeptide. In some embodiments , the receptor targeting sequence directs transport of the receptor to a does not comprise SEQ ID NO : 39 . nucleus, cytoplasm , mitochondria , endoplasmic reticulum In some embodiments, the target polynucleotide is 40 (ER ) , chloroplast , apoplast, or peroxisome. In some embodi genomic DNA . In some embodiments , the target polynucle - ments , the targeting sequence comprises a nuclear export otide is RNA . In some embodiments , the modification is signal (NES ) . In some embodiments , the targeting sequence phosphorylation . comprises plasma membrane targeting peptide . In some In some embodiments, the actuator moiety is a Cas embodiments , the receptor is linked to a polypeptide folding protein that forms a complex with a guide RNA . In some 45 domain . embodiments , the actuator moiety is an RNA binding pro - In an aspect, the present disclosure provides a method of tein (RBP ) complexed with a guide RNA that forms a regulating expression of a target polynucleotide in a cell complex with a Cas protein . In some embodiments , the Cas comprising a nucleus . In some embodiments, the method protein substantially lacks DNA cleavage activity . comprises ( a ) exposing a chimeric intracellular receptor to In some embodiments, ( i ) the GMP forms a portion of the 50 an antigen , wherein ( i ) the receptor comprises an antigen chimeric adaptor polypeptide , ( ii ) the chimeric adaptor poly - interacting domain and actuator moiety , and ( ii ) the receptor peptide is released from the receptor following cleavage of is modified upon exposure to the antigen ; ( b ) translocating the cleavage recognition site , and ( iii ) a further chimeric the modified receptor to the nucleus ; and ( c ) forming a adaptor polypeptide comprising an GMP binds the modified complex between the actuator moiety and the target poly receptor. 55 . In some embodiments , receptor modification comprises In some embodiments , the actuator moiety is a Cas modification at multiple modification sites , and each modi- protein that forms a complex with a guide RNA . In some fication site is effective to bind a chimeric adaptor polypep embodiments , the Cas protein substantially lacks DNA tide . cleavage activity . In some embodiments , the cleavage recognition site com - 60 In some embodiments, the actuator moiety regulates prises a polypeptide sequence, and the cleavage moiety expression of the target polynucleotide by physical obstruc comprises protease activity . In some embodiments , the tion of the target polynucleotide or recruitment of additional cleavage recognition site comprises a disulfide bond , and the factors effective to suppress or enhance expression of the cleavage moiety comprises oxidoreductase activity . In some target polynucleotide . In some embodiments , the actuator embodiments , the cleavage recognition site comprises a first 65 moiety comprises an activator effective to increase expres portion of an intein sequence that reacts with a second sion of the target polynucleotide . portion of the intein sequence to release the actuator moiety. In some embodiments , the antigen is a hormone. US 9 ,856 ,497 B2 In an aspect, the present disclosure provides a chimeric tide (GMP ) linked to the receptor binding moiety , wherein receptor polypeptide . In some embodiments , a chimeric the GMP comprises an actuator moiety linked to a cleavage receptor polypeptide comprises ( a ) an antigen interacting recognition site ; wherein : (i ) the cleavage recognition site is domain ; and ( b ) a gene modulating polypeptide (GMP ) cleavable by a cleavage moiety in response to receptor comprising an actuator moiety linked to a cleavage recog - 5 binding ; and (ii ) the actuator moiety is operable to complex nition site ; wherein : ( 1 ) the chimeric receptor polypeptide is with a target polynucleotide in response to cleavage of the modified in response to antigen binding; (ii ) the cleavage recognition site is cleaved by a cleavage moiety in response cleavage recognition site . In some embodiments , the actua to modification of the chimeric receptor polypeptide; (iii ) the tor moiety is operable to translocate to a after actuator moiety complexes with a target polynucleotide after 10 cleavage of the cleavage recognition sequence . being cleaved from the chimeric receptor polypeptide at the In some embodiments , the actuator moiety is a Cas cleavage recognition site ; and ( iv ) the chimeric receptor protein that forms a complex with a guide RNA. In some polypeptide does not comprise SEQ ID NO : 39. embodiments , the actuator moiety is an RNA binding pro In some embodiments , the cleavage recognition site is tein (RBP ) optionally complexed with a guide RNA that is flanked by the antigen interacting domain and the actuatortuator 15is able00 to form a complex with a Cas protein . In some moiety . embodiments , the Cas protein substantially lacks DNA In some embodiments , the antigen interacting domain cleavage activity . forms a portion of an extracellular region of the chimeric In some embodiments , the cleavage recognition site com receptor polypeptide , and the GMP forms a portion of an prises a polypeptide sequence that is a recognition sequence intracellular region of the chimeric receptor polypeptide. 20 of a protease . In some embodiments, the cleavage recogni In some embodiments , the actuator moiety translocates to tion site comprises a first portion of an intein sequence that a cell nucleus after cleavage of the cleavage recognition reacts with a second portion of the intein sequence to release sequence . In some embodiments , the chimeric receptor the actuator moiety . In some embodiments , the cleavage polypeptide is a that translocates to a cell recognition site comprises a disulfide bond . nucleus in response to antigen binding . 25 In some embodiments , the actuator moiety regulates In some embodiments , the actuator moiety is a Cas expression of the target polynucleotide by physical obstruc protein that forms a complex with a guide RNA . In some tion of the target polynucleotide or recruitment of additional embodiments , the actuator moiety is an RNA binding pro - factors effective to suppress or enhance expression of the tein (RBP ) optionally complexed with a guide RNA that is target polynucleotide . In some embodiments , the actuator able to form a complex with a Cas protein . In some 30 moiety comprises an activator effective to increase expres embodiments , the Cas protein substantially lacks DNA sion of the target polynucleotide . In some embodiments , the cleavage activity . actuator moiety is linked to at least one nuclear localization In some embodiments , the cleavage recognition site com signal (NLS ). prises a polypeptide sequence that is a recognition sequence In some embodiments , the adaptor polypeptide is linked of a protease . In some embodiments , the cleavage recogni - 35 to at least one targeting sequence which directs transport of tion site comprises a first portion of an intein sequence that the adaptor to a specific region of a cell . In some embodi reacts with a second portion of the intein sequence to release ments , the targeting sequence directs transport of the adaptor the actuator moiety . In some embodiments , the cleavage to nucleus , cytoplasm , mitochondria , endoplasmic reticulum recognition site comprises a disulfide bond . (ER ), chloroplast, apoplast , peroxisome or plasma mem In some embodiments , the receptor is a transmembrane 40 brane . In some embodiments , the targeting sequence com receptor. In some embodiments , the receptor is a nuclear prises a nuclear export signal (NES ) . In some embodiments , receptor. the targeting sequence comprises plasma membrane target In some embodiments , the actuator moiety regulates ing peptide . expression of the target polynucleotide by physical obstruc In an aspect, the present disclosure provides a system for tion of the target polynucleotide or recruitment of additional 45 regulating expression of a target polynucleotide in a cell . In factors effective to suppress or enhance expression of the some embodiments, the system comprises ( a ) a chimeric target polynucleotide . In some embodiments , the actuator receptor polypeptide that is modified upon binding an anti moiety comprises an activator effective to increase expres gen , wherein receptor modification comprises a conforma sion of the target polynucleotide . In some embodiments , the tional change or chemical modification ; ( b ) a chimeric actuator moiety is linked to at least one nuclear localization 50 adaptor polypeptide that binds the receptor in response to the signal (NLS ) . receptor modification ; ( c ) an actuator moiety linked to a In some embodiments , the receptor is linked to at least peptide cleavage domain , wherein upon cleavage of the one targeting sequence which directs transport of the recep - peptide cleavage domain , the actuator moiety is activated to tor to a specific region of a cell. In some embodiments , the complex with a target polynucleotide ; and ( d ) a cleavage targeting sequence directs transport of the receptor to a 55 moiety that cleaves the peptide cleavage domain when in nucleus, cytoplasm , mitochondria , endoplasmic reticulum proximity to the peptide cleavage domain ; wherein : ( i ) the ( ER ) , chloroplast, apoplast, peroxisome or plasma mem - cleavage moiety forms an intracellular portion of the recep brane. In some embodiments , the targeting sequence com - tor, and the actuator moiety linked to the peptide cleavage prises a nuclear export signal (NES ) . In some embodiments , domain forms a portion of the chimeric adaptor polypeptide ; the targeting sequence comprises a plasma membrane tar - 60 ( ii ) the cleavage moiety is complexed with a second adaptor geting peptide . In some embodiments , the receptor is linked polypeptide that binds the receptor in response to the recep to a polypeptide folding domain . tor modification , and the actuator moiety linked to the In an aspect , the present disclosure provides a chimeric peptide cleavage domain forms a portion of the chimeric adaptor polypeptide . In some embodiments , a chimeric adaptor polypeptide ; or ( iii ) the cleavage moiety forms a adaptor polypeptide comprises ( a ) a receptor binding moiety 65 portion of the adaptor polypeptide, and the actuator moiety that binds a receptor that has undergone modification upon linked to peptide cleavage domain forms an intracellular binding to an antigen ; and ( b ) a gene modulating polypep portion of the receptor. US 9 ,856 , 497 B2 In an aspect, the present disclosure provides a system for erence to the same extent as if each individual publication , regulating expression of a target polynucleotide in a cell . In patent, or patent application was specifically and individu some embodiments , the system comprises (a ) a chimeric a lly indicated to be incorporated by reference . receptor polypeptide receptor that is modified upon binding an antigen , wherein receptor modification comprises a con - 5 BRIEF DESCRIPTION OF THE DRAWINGS formational change or chemical modification ; (b ) an chime ric adaptor polypeptide that binds the receptor in response to The novel features of the invention are set forth with the receptor modification ; ( c ) an actuator moiety linked to a particularity in the appended claims. A better understanding peptide cleavage domain , wherein upon cleavage of the peptide cleavage domain , the actuator moiety is activated to of the features and advantages of the present invention will complex with a target polynucleotide ; and ( d ) a recombinant 10 be obtained by reference to the following detailed descrip protease domain that cleaves the peptide cleavage domain tion that sets forth illustrative embodiments , in which the when in proximity to the peptide cleavage domain ; wherein : principles of the invention are utilized , and the accompany ( i) the recombinant protease domain forms an intracellular ing drawings of which : portion of the receptor, and the actuator moiety linked to the FIG . 1 shows an exemplary chimeric receptor polypeptide peptide cleavage domain forms a portion of the chimeric 15 comprising an antigen interacting domain and a gene modu adaptor polypeptide ; ( ii ) the recombinant protease domain is lating polypeptide (GMP ). complexed with a second adaptor polypeptide that binds the FIG . 2 shows an exemplary chimeric transmembrane receptor in response to the receptor modification , and the receptor polypeptide . actuator moiety linked to the peptide cleavage domain forms FIG . 3A shows an exemplary chimeric receptor polypep a portion of the chimeric adaptor polypeptide ; or ( iii ) the 20 tide including an actuator moiety comprising an RNA recombinant protease domain forms a portion of the chime- binding protein optionally complexed to a guide nucleic acid ric adaptor polypeptide, and the actuator moiety linked to the ( e . g ., SgRNA ) . FIG . 3B shows an exemplary system com peptide cleavage domain forms an intracellular portion of prising a chimeric receptor polypeptide and a chimeric the receptor. adaptor polypeptide comprising a cleavage moiety . In an aspect, the present disclosure provides a chimeric 25 FIGS . 4A - D illustrate schematically the release of an receptor polypeptide . The chimeric receptor polypeptide actuator moiety from a GMP in a system comprising a comprises : an extracellular antigen interacting domain which binds an antigen ; a transmembrane domain ; and an receptor which undergoes phosphorylation ; FIGS. 4E - H intracellular gene modulation domain comprising a Cas illustrate schematically the release of an actuator moiety protein , wherein a peptide cleavage domain is located at the from a GMP in a system comprising a receptor which amino terminus of the gene modulation domain ; wherein »30 undergoes a conformational change . upon binding of the extracellular antigen interacting domain FIG . 5 shows an exemplary chimeric receptor polypeptide to the antigen , the gene modulation domain is released from comprising at least one targeting sequence . the chimeric receptor polypeptide by cleavage of the peptide FIG . 6A shows an exemplary chimeric adaptor polypep cleavage domain . In some embodiments , the chimeric recep tide comprising a receptor binding moiety and a gene tor polypeptide undergoes a receptor modification upon 35 modulating polypeptide (GMP ). FIG . 6B shows an exem binding to the antigen . In some embodiments , the transmem plary chimeric adaptor polypeptide including an actuator brane domain comprises a portion of a Notch receptor moiety comprising an RNA -binding protein optionally com protein , or any derivative, variant, or fragment thereof. In plexed to a guide nucleic acid (e .g ., SgRNA ). some embodiments , trasmembrane domain comprises an FIG . 7 shows an exemplary system comprising a chimeric sequence having at least 80 % , 81 % , 82 % , 83 % , 40 receptor polypeptide comprising a cleavage moiety and a 84 % , 85 % , 86 % , 87 % , 88 % , 89 % 90 % , 91 % 92 % , 93 % , chimeric adaptor polypeptide comprising a GMP. 94 % , 95 % , 96 % , 97 % , 98 % , 99 % , or 100 % identity to SEQ F IGS. 8A - D illustrate schematically the release of an ID NO : 39 or a fragment thereof. In some embodiments , the actuator moiety from a GMP in a system comprising a Cas protein substantially lacks DNA cleavage activity . In receptor which undergoes phosphorylation ; FIGS. 8E - H some embodiments , the Cas protein is a Cas9 protein . 45 illustrate schematically the release of an actuator moiety In some embodiments , the gene modulation domain fur ther comprises an activator domain effective to increase from a GMP in a system comprising a receptor which expression of a target polynucleotide . In some embodiments, undergoes a conformational change . the gene modulation domain further comprises a FIG . 9 shows an exemplary system comprising a chimeric domain effective to decrease expression of a target poly receptor polypeptide , a chimeric adaptor polypeptide com nucleotide . 50 prising a GMP, and a second adaptor polypeptide compris In some embodiments, the gene modulation domain fur- ing a cleavage moiety . ther comprises at least one targeting sequence which directs FIGS . 10A - D illustrate schematically the release of an transport of the gene modulation domain to a specific region actuator moiety from a GMP in a system comprising at least of a cell after the gene modulation domain is released from two adaptor polypeptides and a receptor which undergoes the receptor. In some embodiments, the at least one targeting 55 phosphorylation ; FIGS . 10E - H illustrate schematically the sequences comprises a nuclear localization sequence (NLS ) . release of an actuator moiety from a GMP in a system In some embodiments , the receptor is linked to at least comprising at least two adaptor polypeptides and a receptor one targeting sequence which directs transport of the recep - which undergoes a conformational change . tor to a specific region of a cell. In some embodiments , the FIG . 11 shows an exemplary chimeric adaptor polypep receptor is linked to a polypeptide folding domain . 60 tide comprising at least one targeting sequence . In some embodiments , the peptide cleavage domain com - FIGS . 12A - C illustrate schematically a system compris prises a recognition sequence of a protease . ing an exemplary intracellular receptor . FIGS. 13A - D illustrate schematically a system in which INCORPORATION BY REFERENCE the cleavage recognition site comprises an intein sequence ; 65 FIGS . 13E - H illustrate an alternative arrangement of a All publications, patents , and patent applications men - system in which the cleavage recognition site comprises an tioned in this specification are herein incorporated by ref- intein sequence . US 9 ,856 ,497 B2 10 FIGS . 14A - D illustrate schematically a system in which cells . FIGS. 22D and 22E illustrates the binding specificity the cleavage recognition site comprises a disulfide bond ; of paxillin - TEV for the beta subunit of integrin relative to FIGS . 14E - H illustrate an alternative arrangement of a the alpha subunit system in which the cleavage recognition site comprises a FIGS. 23A and 23B provide an exemplary embodiment of disulfide bond . 5 chimeric GPCR -gene modulating domain polypeptide. FIG . FIG . 15 shows an illustration adapted from FIG . 2 of 23A shows a scheme of target gene regulation by GPCR Makarova , K . S . et al, “ An updated evolutionary classifica tion of CRISPR -Cas systems, ” Nat Rev Microbiol (2015 ) based chimeric antigen receptor- dCas9 activators . FIG . 23B 13 :722 -736 providing architectures of the genomic loci for shows that the CXCR4 - dCas9 polypeptide was responsive subtypes of CRISPR - Cas systems. to CXCL12 and activated the luminescent reporter. FIGS . FIGS . 16A - D illustrate schematically the release of an U 23C and 23D illustrate chimeric GPCR receptors comprising actuator moiety from a GMP in a system comprising at least LPAR1, CXCR4, and hM3D and corresponding activation two adaptor polypeptides . of a fluorescent reporter (GFP ) in the presence of ligand , FIG . 17 shows a schematic diagram of engineered chi B -arrestin -protease , and sgRNA . FIGS. 23E and 23F com meric antigen receptors of the present invention for gene 15 pare levels of transcriptional regulation of a reporter gene modulation such as genome editing and gene regulation . resulting from dCas9 - VPR targeted by sgRNA after release FIG . 18 shows variants of linkers located between the from a chimeric receptor and a TetR - VPR which binds transmembrane domain and the gene modulation domain of directly to the of the reporter gene . the engineered chimeric antigen receptors of the present FIGS . 24A - 24G show exemplary embodiments of modu disclosure . 20 lar chimeric artificial Notch receptors of the present inven FIGS. 19A - 19F show engineered chimeric antigen recep - tion . FIG . 24A shows a wild -type Notch bound to its ligand tors with gene modulation domains and in some cases, their Delta . After the receptor is activated by binding Delta , the associated adaptor- proteases . FIG . 19A depicts such recom - ICD is cleaved by a protease and translocates to the nucleus binant receptors that bind to cell surface . FIG . 19B to regulate target genes. FIG . 24B shows a chimeric artificial depicts recombinant receptors for gene modulation that can 25 Notch receptor where the Notch ICD has been replaced with bind soluble antigens . FIG . 19C illustrates gene modulating , adCas9 fusion protein . The dCas9 fusion protein can engineered receptors that can bind to extracellular matrix include an effector domain such as an activator domain , e . g . , (ECM ) signals . FIG . 19D illustrates dimerizing receptors. VP64 domain or a repressor domain , e . g . , KRAB domain . One of the receptors includes an extracellular domain FIG . 24C shows another chimeric artificial Notch receptor (ECD ), a transmembrane domain ( TM ) , an intracellular 30 containing a dCas9 fusion protein where the Notch ECD has domain ( ICD ) , a peptide- cleavage sequence , and a gene been replaced with a CD47 -binding scFv. FIG . 24D shows modulating effector domain . The other receptor of the dimer an exemplary modular chimeric artificialNotch receptor and includes an ECD , TM , ICD , and a protease . FIG . 19E shows an adapter - protease fusion protein (presinillin - TEV pro another example of dimerizing receptors that can modulate tease ) expressed on the surface of a cell such as an immune gene expression or edit genes . One of the dimerizing recep - 35 cell . The modular chimeric artificial Notch receptor can tors can include an ECD , TM , ICD , peptide -cleavage contain a dCas9 fusion polypeptide , a linker, and an effector sequence , and a genemodulating effector domain . The other domain . Upon Delta - Notch binding , the presinillin can asso receptor of the dimer can include an ECD , TM and ICD , and ciate with the chimeric artificial Notch receptor . Then , the not a protease . The protease that cleaves this dimerizing TEV protease can cleave the peptide cleavage domain of the receptor can be fused to an adaptor protein that associates to 40 chimeric artificialNotch receptor. Activation of a fluorescent the activated dimerizing receptor . FIG . 19F shows an reporter in cells expressing the Notch - dCas9 - activator is example of an oligomerizing receptor that includes engi shown in FIG . 24E for HEK293 cells , in FIG . 24F for Jurkat neered chimeric antigen receptors fused to gene modulation cells , and in FIG . 24G in THP - 1 macrophages . domains. FIGS . 25A - 25C show theoretical models for reshaping FIGS. 20A and 20B provides different chimeric antigen 45 the endogenous response of the Notch receptor using the receptors and illustrates the binding of a dCas9 - activator chimeric antigen Notch receptors described herein . FIG . domain guided to a target gene by an sgRNA . FIG . 20A 25A shows the repression of the endogenous phagocytic shows recombinant chimeric antigen receptor polypeptides response of a receiving cell expressing endogenous Notch and in some cases, their associated adaptor- protease poly - upon binding Delta expressed on a signaling cell . FIG . 25B such as Notch and presenillin -proteases , GPCRs 50 shows that the engineered can receptor can be created to and B2 - arrestin -proteases , and paxillin -proteases , rewire the endogenous phagocytic response to an external cadherins and B -catenin -proteases , death receptors and Delta signal. FIG . 25C shows that the engineered can FADD - proteases , and chimeric antigen receptors . receptor can be produced to shift the repression of the cell ' s FIG . 21A show uses of chimeric antigen GPCRs coupled endogenous phagocytic response to activation upon Delta to dCas9 - activators . FIG . 21B illustrates an alternative con - 55 binding . figuration in which the protease moiety is coupled to the FIGS. 26A -26D show that the Notch -dCas9 activator , GPCR and the dCas9 -activator domain is coupled to an upon Delta binding , can activate target genes such as those adaptor protein recruited to an activated GPCR . that control cell or the cell cycle . Cells expressing FIGS . 22A and 22B show integrin - dCas9 gene modulat - the Notch - dCas9 polypeptide activated the target genes ing polypeptides and their response to integrin ligands such 60 when in the presence of Delta (FIGS . 26B and 26D ). Notch as fibronectin . FIG . 22A depicts a schematic diagram of a chimeric antigen receptor did not activate transcription of chimeric antigen integrin - dCas9 activator. FIG . 22B shows the target genes in the absence of Delta ( FIGS . 26A and an integrin -dCas9 complex that is responsive to fibronectin . 26C ). Upon binding to an sgRNA specific to the reporter gene , the FIGS. 27A and 27B show that the Notch - dCas9 activator recombinant complex activated transcription of the reporter 65 is guided by a sgUAS (SEQ ID NO : 1 ; gtactccgacctctagtgt ) (H2B -GFP ) . FIG . 22C illustrates the activity of integrin - to a UAS promoter and activates transcription of a reporter dCas9 complex in adherent cells compared to suspension gene (H2B -citrine ). FIG . 27A provides a schematic diagram US 9 , 856 , 497 B2 12 of the process. FIG . 27B shows that the Notch -dCas9 expression ) in CHO UAS -H2B clones stably selected for activator is responsive to Delta . NC5 ( S . pyogenes dCas9 , and sgUAS ) or in CHO CSL -H2B FIGS. 28A and 28B show that the CXCR4- dCas9 - VPR clones with wild - type human Notchl or NC5 ( S . aureus polypeptide is responsive to CXCL12 ligand and activates dCas9, and sasgCSL ) and cultured on bare or immobilized transcription of a reporter gene ( luciferase ). FIG . 28A pro - 5 Delta surfaces for 4 days (n = 3 ) . Mean : SEM . FIG . 31F vides a schematic diagram of ligand binding of the CXCR4 - shows percentage of cells that activate H2B - citrine in CHO dCas9 - VPR polypeptide that is complexed with a sgRNA UAS -H2B clones stably selected for NC5 ( S . pyogenes ( sgTET ; SEQ ID NO : 2 ; gtacgttctctatcactgata ). FIG . 28A also dCas9 , and sgUAS ) or in CHO 12xCSL -H2B clones with shows a ß - arrestin - protease fusion protein that can associ- wild - type human Notch1 or NC5 ( S . aureus dCas9 , and ated with the engineered chimeric antigen receptor. The 10 sasgCSL ) and cultured on bare or immobilized -Delta sur diagram also shows ( 1 ) translocation of free dCas9 - VPR faces for 4 days ( n = 3 ) . Mean?SEM , * * p < 0 .01 , compared to into the nucleus , ( 2 ) binding of the sgTET- dCas9 -VPR ( - Delta ) controls. FIG . 31G illustrates activation of NC5 complex to a Teto promoter that regulates transcription of receptors by immobilized Delta ligand resulting in cleavage the luciferase gene, and ( 3 ) transcription of the reporter . FIG . and nuclear translocation of dCas9 -VPR . FIG . 31H shows 28B shows that transcription of luciferase gene is regulated 15 EGFP reporter intensity historgrams from HEK293T by CXCL2 binding to the CXCR4 -dCas9 - VPR polypeptide . reporter cells stably expressing a tet - inducible EGFP gene FIGS . 29A and 29B show that the integrin - dCas9 -VPR and a targeting sgRNA (sgTET ) in the presence of dCas9 polypeptide is responsive to an extracellular matrix ligand VPR , NC5 + Delta + DAPT, NC5 + Delta , NC5 -Delta , and no and activates transcription of a reporter gene (H2B - citrine ). construct. FIG . 311 shows contour plots (where the same FIG . 29A shows a schematic diagram of transcriptional 20 number of cells fall between each pair of contour lines ) of activation of the reporter upon ligand binding to the integrin - EGFP activation of HEK293T reporter cells transfected with dCas9 - VPR polypeptide . FIG . 29B shows that the integrin NC5 receptor and cultured on various concentrations of based engineered chimeric antigen receptor - dCas9 complex immobilized Delta for 3 days . induced reporter expression in response to signals from the FIG . 32A illustrates a schematic of a synthetic Cas9 ??? . 25 receptor system in accordance with an embodiment FIGS . 30A and 30B show exemplary embodiments of the described herein . Modulation of target endogenous genes is chimeric antigen receptor- effector polypeptides described responsive to extracellular signals , such as Delta . A variety herein that are based on a " Split AND gate ” or “ Cascade of downstream cellular behaviors can be triggered from AND gate ” logic . FIG . 30A shows a schematic diagram of native extracellular inputs , depending on design of the a split dCas9 effector tethered to separate engineered recep - 30 system . FIG . 32B left shows CDKN1B activation via tors. FIG . 30B provides a schematic diagram of a chimeric h NECD -dCas9 - VPR ( construct NC5 ) induces a Delta -de receptor- tTa polypeptide that, upon binding to its ligand , pendent cell- cycle arrest at G0 /G1 phase . Right shows a induces expression of a Teto - driven chimeric receptor- schematic representation of Delta - dependent cleavage of dCas9 polypeptide . dCas9 - VPR from NC5 leading to CDKN1B - high and FIGS . 31A -311 illustrate embodiments of a receptor- 35 GO /G1 - arrested cells (with 2n DNA ) , in accordance with an based strategy to mobilize Cas9 in response to extracellular embodiment. FIG . 32C shows example flow -cytometry plots signals . FIG . 31A illustrates activation of Notch1 receptors and percentage quantification ( shaded regions ) of involving cleavage and nuclear translocation of the Notch CDKN1B -high and GO/ G1 - arrested cells under different intracellular domain , which can be replaced by or engi - culture conditions: HEK293 cells with SgRNA only , neeredne to promote expression of Cas9 derivatives . The 40 sgRNA + Delta , sgRNA + NC5, or sgRNA + NC5 + Delta . NC5 fusion of effector domains to Cas9 and a user -defined was transiently transfected in sgCDKN1B - integrated cells , single - guide RNA ( SORNA ) sequence allow for targeted which were then cultured on Delta - coated or bare surfaces gene regulation . FIG . 31B shows schematic designs of for 4 days . n = 3 , Mean : SEM . GO /G1 arrested cells corre mCherry - tagged chimeric receptor constructs that were ini spond to the top left shaded corner of the plots, and the top tially tested for cellular localization and Delta - dependent 45 bar of each pair in the bar graph . G2 arrested cells corre reporter activation . The human codon - optimized nuclease spond to the top right shaded corner of the plots , and bottom dead Cas9 (dCas9 ) and tripartite activator domains (VP64 , bar of each pair in the bar graph . p65 , and Rta ; VPR ) are fused immediately after the Notch1 FIG . 33 left panel shows an example confocal fluores extracellular domain (hNECD ) and transmembrane domain . cence micrograph of a CHO cell transfected with construct Construct NC5 comprises maturation signals derived from a 50 NC1 ( ) and sgUAS (blue ) . Premature activation of H2B known ER export signal. FIG . 31C shows a schematic of a (green , nucleus ) is observed . Right panel shows a schematic Chinese hamster ovary (CHO ) cell line integrated with an representation of balance between the strengths of nuclear Upstream Activating Sequence (UAS ) or CSL - binding ( not localization signals and membrane maturation signals in shown ) promoters that drive a Histone 2B (H2B ) - citrine Delta - dependent cleavage of Notch - Cas9 chimeras . reporter gene and a stably integrated promoter- targeting 55 FIG . 34 shows fluorescence microscopy images of CHO SgRNA ( e . g . , SGUAS or sasgCSL , respectively ) used to cells transfected with constructs NC1- NC4 ( top , see FIG . validate gene -activation efficiency of chimeric receptors 31B ) and having H2B expression (bottom ) in the absence of when cultured with or without surface - immobilized Delta . Delta . Scale bar, 20 um . Activation of NC5 receptors by immobilized Delta ligands FIG . 35A shows a naturally occurring NLS sequence in S . leads to cleavage and nuclear translocation of dCas9 - VPR . 60 py . Cas9 found at amino acid residues 647 -670 . The illus dCas9 - VPR complexed with a sequence - specific sgRNA tration shows this “ intrinsic ’ NLS ( ?NLS ) can form a surface ( e . g . , SGUAS or sasgCSL ) allows for binding of the complex accessible helix - linker- helix structure (PDB ID : 4UN3 ) to the promoter and activation of H2B -citrine gene. FIG . (SEQ ID NO : 33 , 34 ) . FIG . 35B shows example results for 31D shows example microscopy images of CHO cells HEK293 cells stably integrated with pTET- EGFP reporter transfected with the NC5 chimera resulting in H2B expres - 65 and targeting sgRNA (sgTET ) that were transfected with sion when exposed to immobilized Delta for 4 days. Scale d Cas9 - VPR ( top ) or mutated - iNLS dCas9 - VPR (center ) . bar, 20 um . FIG . 31E shows relative H2B levels ( normalized Representative histograms of EGFP activation in the pres US 9 , 856 , 497 B2 13 14 ence or absence (bottom ) of constructs and quantification of Molecular Biology ( F . M . Ausubel , et al. eds. ) ; the series EGFP activation (normalized to untransfected cells ) is Methods In Enzymology (Academic Press , Inc . ), PCR 2 : A shown (SEQ ID NO : 31, 32 ). FIG . 35C shows by micros Practical Approach (M . J . MacPherson , B . D . Hames and G . copy impaired nuclear localization and EGFP reporter acti - R . Taylor eds. ( 1995 ) ) , Harlow and Lane , eds . ( 1988 ) vation in HEK293T cells when the iNLS motif is disrupted 5 , A Laboratory Manual, and Culture of Animal ( SEQ ID NO : 31 , 32 , 9 ) . FIGS. 35C and 35D show visually Cells: A Manual of Basic Technique and Specialized Appli by microscopy and quantitatively that adding a synthetic cations , 6th Edition ( R . I . Freshney , ed . ( 2010 ) ) . NLS to the N - terminus of iNLS -mutated dCas9 - VPR par As used in the specification and claims, the singular forms tially restores EGFP activation . " a " , " an " and " the " include plural references unless the FIGS. 36A - 36E show Delta -dependent gene editing. FIG . 10 context clearly dictates otherwise. For example , the term “ a 36A shows schematically Delta -dependent DNA - cutting chimeric transmembrane receptor ” includes a plurality of with an NC5 variant : hNECD fused to wild -type S . pyogenes chimeric transmembrane receptors . nuclease -active Cas9 ( hNECD -Cas9 ). FIG . 36B shows the The term “ about ” or “ approximately ” means within an of gene editing/ cutting with hNECD - Cas9 in CHO acceptable error range for the particular value as determined cells with stably integrated EGFP and a targeting sgRNA 15 by one of ordinary skill in the art, which will depend in part ( sgEGFP ) . FIG . 36C shows an example schematic for two on how the value is measured or determined , i. e . , the SORNAs (short bars to left and right of " sgCXCR4 ” ) that limitations of the measurement system . For example , were stably expressed in HEK293T cells and were designed " about” can mean within 1 or more than 1 standard devia to target the 5 ' untranslated region (UTR ) and 1 of tion , per the practice in the art . Alternatively, “ about” can CXCR4. Scale bar , 1000 bp . FIG . 36D , top panel, shows 20 mean a range of up to 20 % , up to 10 % , up to 5 % , or up to example results from a T7E1 endonuclease to assay the 1 % of a given value . Alternatively , particularly with respect extent of Delta - induced hNECD - Cas9 -mediated modifica - to biological systems or processes , the term can mean within tion of CXCR4 gene in HEK293T cells , as detected by an order of magnitude , preferably within 5 - fold , and more amount of products cleaved by T7E1 in SDS - PAGE gels . preferably within 2 - fold , of a value . Where particular values The bottom panel shows example results for frequency of 25 are described in the application and claims, unless otherwise CXCR4 indel . FIG . 36E shows example results stated , the term “ about” meaning within an acceptable error for the quantification of flow cytometry -based immunofluo range for the particular value should be assumed . rescence staining of CXCR4 protein expression in As used herein , a " cell” can generally refer to a biological HEK293T cells . cell. A cell can be the basic structural, functional and / or FIG . 37A shows alignment of EGF - 11 and - 12 repeats of 30 biological unit of a living organism . A cell can originate human , Xenopus , , and homologs (SEQ from any organism having one or more cells . Some non ID NO : 57 -60 ) . Identical residues are indicated in gray limiting examples include : a prokaryotic cell , eukaryotic boxes . Asterisk (* ) marks conserved cysteine and Ca2 + cell, a bacterial cell , an archaeal cell , a cell of a single -cell binding consensus residues. FIG . 37B shows variable acti - eukaryotic organism , a protozoa cell, a cell from a plant ( e. g . vation levels of various EGF deletion variants ( as evaluated 35 cells from plant crops, fruits , vegetables , grains, soy bean , by reporter assay ) . corn , maize, wheat , seeds , tomatoes , rice , cassava , sugar FIG . 38 shows the simultaneous activation of CXCR4 and cane , pumpkin , hay , potatoes , cotton , cannabis , tobacco , CD95 genes targeted by gene -specific sgRNAs ( 2 per gene ) . flowering plants , conifers , gymnosperms, ferns, clubmosses , Relative activation levels of CXCR4 and CD95 under indi- hornworts , liverworts , mosses ) , an algal cell, ( e . g . , Botryo cated conditions in 4 -day cultures are shown (a .u ., arbitrary 40 coccus braunii , Chlamydomonas reinhardtii , Nannochlo units ) . Data is displayed as mean fluorescence ropsis gaditana , Chlorella pyrenoidosa , Sargassum patens intensity = SEM ( n = 3 independent experiments ) . # ,# # p < 0 .01 , C . Agardh , and the like ), seaweeds (e . g . kelp ) , a fungal cell 0 .001 , *, * * p < 0 .01 , 0 .001 , compared to CXCR4 and CD95 ( e .g . , a yeast cell, a cell from a mushroom ) , an animal cell, levels, respectively in negative control, dCas9 - VPR + SONT a cell from an invertebrate animal ( e . g . fruit fly , cnidarian , ( non - targeting ) . 45 echinoderm , nematode , etc . ) , a cell from a vertebrate animal FIGS. 39A -39F show the conversion of Delta signals to (e .g ., fish, amphibian , reptile , bird , ) , a cell from a cell cycle phase - specific arrest . FIG . 39 A illustrates sche - mammal ( e . g . , a pig , a cow , a goat, a , a rodent, a , matically the use ofminimal NC5 receptor variant to elicit a mouse , a non -human primate, a human , etc .) , and etcetera . Delta - dependent arrest of the cell cycle . FIG . 39B depicts Sometimes a cell is not originating from a natural organism cellular arrest at the GO /G1 phase resulting from CDKN1B 50 ( e . g . a cell can be a synthetically made, sometimes termed overexpression . FIGS . 39C and 39D shows that in cells with an artificial cell ) . dCas9 - VPR and sgCDKN1B , CDKN1B upregulation was The term “ nucleotide , ” as used herein , generally refers to concomitant with GO /G1 enrichment , and in cells with a base - sugar -phosphate combination . A nucleotide can com dCas9 - VPR and non - targeting gsRNA , a minimal increase prise a synthetic nucleotide . A nucleotide can comprise a in CDKN1B was observed . FIGS . 39E and 39F show 55 synthetic nucleotide analog. can be monomeric abrogation of Delta - induced CDKN1B upregulation and units of a nucleic acid sequence ( e . g . deoxyribonucleic acid GO /G1 arrest in cells with DAPT . (DNA ) and ribonucleic acid (RNA ) ) . The term nucleotide can include ribonucleoside triphosphates triphos DETAILED DESCRIPTION phate (ATP ) , triphosphate (UTP ) , triphos 60 phate (CTP ) , triphosphate (GTP ) and deoxyribo The practice of some methods disclosed herein employ , nucleoside triphosphates such as dATP , dCTP, dITP , DUTP , unless otherwise indicated , conventional techniques of dGTP , DTTP , or derivatives thereof. Such derivatives can immunology , biochemistry , chemistry , molecular biology, include, for example , [AS ] DATP , 7 -deaza -dGTP and microbiology , cell biology , genomics and recombinant 7 -deaza - dATP , and nucleotide derivatives that confer nucle DNA , which are within the skill of the art. See for example 65 ase resistance on the nucleic acid molecule containing them . Sambrook and Green , Molecular : A Laboratory The term nucleotide as used herein can refer to dideoxyri Manual, 4th Edition ( 2012 ) ; the series Current Protocols in bonucleoside triphosphates ( ddNTPs ) and their derivatives . US 9 , 856 ,497 B2 15 16 Illustrative examples of dideoxyribonucleoside triphos - dihydrouridine, queuosine , and wyosine. Non - limiting phates can include, but are not limited to , ddATP, ddCTP , examples of polynucleotides include coding or non -coding ddGTP , ddITP , and ddTTP . A nucleotide may be unlabeled regions of a gene or gene fragment, loci ( locus ) defined from or detectably labeled by well -known techniques. Labeling linkage analysis , , , messenger RNA (mRNA ) , can also be carried out with quantum dots . Detectable labels 5 transfer RNA (TRNA ), ribosomal RNA (rRNA ), short inter can include , for example, radioactive isotopes , fluorescent fering RNA (siRNA ), short- hairpin RNA ( shRNA ), micro labels, chemiluminescent labels, bioluminescent labels and labels . Fluorescent labels of nucleotides may RNA (miRNA ), ribozymes, cDNA , recombinant polynucle include but are not limited fluorescein , 5 -carboxyfluorescein otides, branched polynucleotides , plasmids, vectors , isolated (FAM ), 2 ' 7 '- dimethoxy - 4 '5 - dichloro - 6 - carboxyfluorescein 10 DNA of any sequence, isolated RNA of any sequence , ( JOE ) , rhodamine , 6 -carboxyrhodamine (R6G ), N , N , N ', N ' cell - free polynucleotides including cell- free DNA ( cfDNA ) tetramethyl- 6 - carboxyrhodamine ( TAMRA ) , 6 - carboxy - X and cell- free RNA (cfRNA ) , nucleic acid probes , and prim rhodamine (ROX ), 4 - (4 ' dimethylaminophenylazo ) benzoic ers . The sequence of nucleotides can be interrupted by acid (DABCYL ) , Cascade Blue, Oregon Green , Texas Red , non -nucleotide components . Cyanine and 5 - ( 2 - aminoethyl) aminonaphthalenealene - 1- - sulfonic 15 The terms “ target polynucleotide” and “ target nucleic acid (EDANS ) . Specific examples of fluorescently labeled acid ,” as used herein , refer to a nucleic acid or polynucle nucleotides can include [R6G ]dUTP , TAMRAZUTP, otide which is targeted by an actuator moiety of the present [ R110 ] dCTP, [R6G ] dCTP, [TAMRA ] dCTP, [ JOE ] ddATP , disclosure . A target nucleic acid can be DNA . A target [R6G ] ddATP, [FAM ] ddCTP, [R110 ] ddCTP , [ TAMRA ] nucleic acid can be RNA . A target nucleic acid can refer to ddGTP , FROX ]ddTTP , FdR6G ] ddATP , [ dR110 ] ddCTP , 20 a chromosomal sequence or an extrachromosomal sequence , [DTAMRA ]ddGTP , and [DROX ]ddTTP available from Per ( e. g ., an episomal sequence , a minicircle sequence , a mito kin Elmer, Foster City , Calif . FluoroLink DeoxyNucle chondrial sequence , a chloroplast sequence , etc . ) . A target otides, FluoroLink Cy3 -dCTP , FluoroLink Cy5 -dCTP , Fluo nucleic acid can be a nucleic acid sequence thatmay not be roLink Fluor X - DCTP, FluoroLink Cy3 - dUTP , and related to any other sequence in a nucleic acid sample by a FluoroLink Cy5 -dUTP available from Amersham , Arlington 25 single nucleotide substitution . A target nucleic acid can be a Heights , Ill. ; Fluorescein - 15 - dATP , Fluorescein - 12 - dUTP , nucleic acid sequence that may not be related to any other Tetramethyl - rodamine - 6 -DUTP , IR770 - 9 -DATP , Fluores sequence in a nucleic acid sample by a 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , cein - 12 - ddUTP , Fluorescein - 12 -UTP , and Fluorescein - 15 or 10 nucleotide substitutions . In some embodiments , the 2 '- DATP available from Boehringer Mannheim , Indianapo substitution may not occur within 5 , 10 , 15 , 20 , 25 , 30 , or 35 lis , Ind . ; and Labeled Nucleotides , BODIPY - 30 nucleotides of the 5 ' end of a target nucleic acid . In some FL - 14 -UTP , BODIPY - FL - 4 -UTP , BODIPY - TMR - 14 -UTP , embodiments , the substitution may not occur within 5 , 10 , BODIPY - TMR - 14 - dUTP , BODIPY - TR - 14 -UTP , BODIPY 15 , 20 , 25 , 30 , 35 nucleotides of the 3' end of a target nucleic TR - 14 - dUTP, Cascade Blue - 7 -UTP , Cascade Blue - 7 -DUTP , acid . In general, the term “ target sequence ” refers to a fluorescein - 12 -UTP , fluorescein - 12 - dUTP , Oregon Green nucleic acid sequence on a single strand of a target nucleic 488 - 5 - DUTP , Rhodamine Green - 5 -UTP , Rhodamine Green - 35 acid . The target sequence can be a portion of a gene , a 5 - DUTP , tetramethylrhodamine -6 -UTP , tetramethylrhod regulatory sequence , genomic DNA , cell free nucleic acid amine- 6 - dUTP , Texas Red -5 -UTP , Texas Red - 5 - dUTP , and including cfDNA and/ or cfRNA , DNA , a fusion gene , and Texas Red - 12 - dUTP available from Molecular Probes , RNA including mRNA , miRNA , rRNA , and others . Eugene, Oreg . Nucleotides can also be labeled or marked by The term " gene, ” as used herein , refers to a nucleic acid chemical modification . A chemically -modified single 40 (e .g ., DNA such as genomic DNA and cDNA ) and its nucleotide can be biotin - dNTP . Some non - limiting examples corresponding nucleotide sequence that is involved in of biotinylated dNTPs can include , biotin - dATP ( e . g ., bio encoding an RNA transcript. The term as used herein with N6 -ddATP , biotin - 14 -dATP ) , biotin - dCTP ( e . g . , biotin - 11 - reference to genomic DNA includes intervening , non - coding CTP, biotin - 14 -dCTP ), and biotin - dUTP (e . g . biotin - 11 regions as well as regulatory regions and can include 5 ' and DUTP , biotin - 16 - DUTP , biotin - 20 - dUTP ). 45 3 ' ends . In some uses , the term encompasses the transcribed The terms " polynucleotide, ” " , " and sequences, including 5 ' and 3 ' untranslated regions ( 5 ' -UTR “ nucleic acid ” are used interchangeably to refer to a poly - and 3 ' -UTR ), exons and introns . In some genes, the tran meric form of nucleotides of any length , either deoxyribo - scribed region will contain " open reading frames ” that nucleotides or ribonucleotides , or analogs thereof, either in encode polypeptides . In some uses of the term , a " gene ” single - , double - , or multi - stranded form . A polynucleotide 50 comprises only the coding sequences ( e . g . , an “ open reading can be exogenous or endogenous to a cell . A polynucleotide frame” or “ coding region ” ) necessary for encoding a poly can exist in a cell - free environment. A polynucleotide can be peptide. In some cases, genes do not encode a polypeptide , a gene or fragment thereof. A polynucleotide can be DNA . for example , ribosomal RNA genes (rRNA ) and transfer A polynucleotide can be RNA . A polynucleotide can have RNA ( TRNA ) genes . In some cases, the term “ gene ” includes any three dimensional structure , and can perform any func - 55 not only the transcribed sequences , but in addition , also tion , known or unknown . A polynucleotide can comprise one includes non - transcribed regions including upstream and or more analogs ( e . g . altered backbone , sugar, or nucle downstream regulatory regions , enhancers and promoters . A obase ) . If present, modifications to the nucleotide structure gene can refer to an “ endogenous gene” or a native gene in can be imparted before or after assembly of the polymer. its natural location in the genomeof an organism . A gene can Some non - limiting examples of analogs include : 5 -bromou - 60 refer to an " exogenous gene ” or a non - native gene . A racil , peptide nucleic acid , xeno nucleic acid , morpholinos, non - native gene can refer to a gene not normally found in the locked nucleic acids, glycol nucleic acids, threose nucleic host organism but which is introduced into the host organism acids, dideoxynucleotides, cordycepin , 7 -deaza -GTP , floro by gene transfer. A non -native gene can also refer to a gene phores (e . g . rhodamine or flurescein linked to the sugar ), not in its natural location in the genome of an organism . A thiol containing nucleotides, biotin linked nucleotides , fluo - 65 non -native gene can also refer to a naturally occurring rescent base analogs, CpG islands, methyl - 7 - guanosine , nucleic acid or polypeptide sequence that comprises muta methylated nucleotides, , thiouridine, pseudourdine , tions, insertions and /or deletions (e . g. , non -native sequence ). US 9 ,856 , 497 B2 17 18 The terms “ transfection ” or “ transfected ” refer to intro sequence , 5 and 3 ' untranslated regions, or an intronic duction of a nucleic acid into a cell by non - viral or viral sequence , which are involved in transcriptional regulation . based methods . The nucleic acid molecules may be gene These cis - acting sequences typically interact with proteins sequences encoding complete proteins or functional portions or other biomolecules to carry out ( turn on / off , regulate , thereof. See , e . g ., Sambrook et al. , 1989 , Molecular Cloning: 5 modulate , etc . ) gene transcription . A " constitutive promoter " A Laboratory Manual, 18 . 1 - 18 . 88 . is one that is capable of initiating transcription in nearly all The term " expression ” refers to one or more processes by tissue types, whereas a " tissue -specific promoter ” initiates which a polynucleotide is transcribed from a DNA template transcription only in one or a few particular tissue types . An ( such as into an mRNA or other RNA transcript) and /or the " inducible promoter " is one that initiates transcription only process by which a transcribed mRNA is subsequently 10 under particular environmental conditions , developmental translated into peptides , polypeptides , or proteins. Tran - conditions, or drug or chemical conditions. scripts and encoded polypeptides can be collectively The terms “ complement, ” “ complements ," " complemen referred to as “ gene product. " If the polynucleotide is tary , ” and “ complementarity , " as used herein , generally refer derived from genomic DNA , expression can include splicing to a sequence that is fully complementary to and hybridiz of the mRNA in a eukaryotic cell. “ Up - regulated , ” with 15 able to the given sequence . In some cases, a sequence reference to expression , generally refers to an increased hybridized with a given nucleic acid is referred to as the expression level of a polynucleotide (e . g. , RNA such as " complement” or “ reverse- complement ” of the given mol mRNA ) and / or polypeptide sequence relative to its expres - ecule if its sequence of bases over a given region is capable sion level in a wild - type state while " down - regulated ” of complementarily binding those of its binding partner, generally refers to a decreased expression level of a poly - 20 such that , for example , A - T , A - U , G - C , and G - U base pairs nucleotide ( e . g ., RNA such as mRNA ) and / or polypeptide are formed . In general, a first sequence that is hybridizable sequence relative to its expression in a wild -type state . to a second sequence is specifically or selectively hybridiz Expression of a transfected gene can occur transiently or able to the second sequence , such that hybridization to the stably in a cell. During “ transient expression ” the transfected second sequence or set of second sequences is preferred ( e . g . gene is not transferred to the daughter cell during cell 25 thermodynamically more stable under a given set of condi division . Since its expression is restricted to the transfected tions, such as stringent conditions commonly used in the art ) cell, expression of the gene is lost over time. In contrast, to hybridization with non - target sequences during a hybrid stable expression of a transfected gene can occur when the ization reaction . Typically , hybridizable sequences share a gene is co - transfected with another gene that confers a degree of sequence complementarity over all or a portion of selection advantage to the transfected cell . Such a selection 30 their respective lengths , such as between 25 % - 100 % advantage may be a resistance towards a certain toxin that is complementarity , including at least 25 % , 30 % , 35 % , 40 % , presented to the cell . 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , The term " expression cassette ,” “ expression construct, ” 91 % 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , 99 % , and or “ expression vector” refers to a nucleic acid that includes 100 % sequence complementarity . Sequence identity , such as a nucleotide sequence such as a coding sequence and a 35 for the purpose of assessing percent complementarity , can be template sequence , and sequences necessary for expression measured by any suitable alignment algorithm , including but of the coding sequence . The expression cassette can be viral not limited to the Needleman -Wunsch algorithm ( see e . g . the or non - viral. For instance , an expression cassette includes a EMBOSS Needle aligner available at www .ebi . ac .uk / Tools / nucleic acid construct , which when introduced into a host psalemboss _ needle / nucleotide. html , optionally with default cell, results in transcription and / or of a RNA or 40 settings ) , the BLAST algorithm ( see e . g . the BLAST align polypeptide , respectively . Antisense constructs or sense con - ment tool available at blast . ncbi. nlm .nih . gov / Blast .cgi , structs that are not or cannot be translated are expressly optionally with default settings ), or the Smith - Waterman included by this definition . One of skill will recognize that algorithm ( see e . g . the EMBOSS Water aligner available at the inserted polynucleotide sequence need not be identical, www . ebi. ac . uk / Tools /psa / emboss _ water /nucleotide .html , but may be only substantially similar to a sequence of the 45 optionally with default settings ) . Optimal alignment can be gene from which it was derived assessed using any suitable parameters of a chosen algo A " plasmid , " as used herein , generally refers to a non - rithm , including default parameters . viral expression vector, e . g . , a nucleic acid molecule that Complementarity can be perfect or substantial /sufficient . encodes for genes and /or regulatory elements necessary for Perfect complementarity between two nucleic acids can the expression of genes . A " viral vector, " as used herein , 50 mean that the two nucleic acids can form a duplex in which generally refers to a viral -derived nucleic acid that is capable every base in the duplex is bonded to a complementary base of transporting another nucleic acid into a cell . A viral vector by Watson -Crick pairing . Substantial or sufficient comple is capable of directing expression of a protein or proteins mentary can mean that a sequence in one strand is not encoded by one or more genes carried by the vector when it completely and /or perfectly complementary to a sequence in is present in the appropriate environment. Examples for viral 55 an opposing strand , but that sufficient bonding occurs vectors include , but are not limited to retroviral, adenoviral, between bases on the two strands to form a stable hybrid lentiviral and adeno - associated viral vectors . complex in set of hybridization conditions ( e . g ., salt con The term " promoter, " as used herein , refers to a poly - centration and temperature ) . Such conditions can be pre nucleotide sequence capable of driving transcription of a dicted by using the sequences and standard mathematical coding sequence in a cell. Thus, promoters used in the 60 calculations to predict the Tm of hybridized strands, or by polynucleotide constructs of the disclosure include cis - empirical determination of Tm by using routine methods . acting transcriptional control elements and regulatory The terms “ peptide, " " polypeptide ,” and “ protein ” are sequences that are involved in regulating or modulating the used interchangeably herein to refer to a polymer of at least timing and /or rate of transcription of a gene. For example , a two amino acid residues joined by peptide bond (s ). This promoter can be a cis - acting transcriptional control element, 65 term does not connote a specific length of polymer , nor is it including an enhancer, a promoter, a transcription termina - intended to imply or distinguish whether the peptide is tor, an origin of replication , a chromosomal integration produced using recombinant techniques , chemical or enzy US 9 ,856 ,497 B2 19 20 matic synthesis , or is naturally occurring . The terms apply to transcription level , for example , by regulating the produc naturally occurring amino acid polymers as well as amino tion of mRNA from DNA , such as chromosomal DNA or acid polymers comprising at least one modified amino acid . cDNA . In some embodiments , an actuator moiety recruits at In some cases, the polymer can be interrupted by non - amino least one that binds to a specific DNA acids . The terms include amino acid chains of any length , 5 sequence , thereby controlling the rate of transcription of including full length proteins , and proteins with or without genetic information from DNA to mRNA . An actuator secondary and/ or tertiary structure ( e . g . , domains ). The moiety can itself bind to DNA and regulate transcription by terms also encompass an amino acid polymer that has been physical obstruction , for example preventing proteins such modified , for example , by disulfide bond formation , glyco - as RNA polymerase and other associated proteins from sylation , lipidation , acetylation , phosphorylation , oxidation , 10 assembling on a DNA template . An actuator moiety can and any other manipulation such as conjugation with a regulate expression of a gene at the translation level, for labeling component. The terms " amino acid ” and “ amino example , by regulating the production of protein from acids , " as used herein , generally refer to natural and non - mRNA template . In some embodiments , an actuator moiety natural amino acids , including, but not limited to , modified regulates gene expression by affecting the stability of an amino acids and amino acid analogues . Modified amino 15 mRNA transcript. In some embodiments , an actuator moiety acids can include natural amino acids and non -natural amino regulates expression of a gene by editing a nucleic acid acids , which have been chemically modified to include a sequence ( e . g . , a region of a genome ). In some embodi group or a chemical moiety not naturally present on the ments , an actuator moiety regulates expression of a gene by amino acid . Amino acid analogues can refer to amino acid editing an mRNA template . Editing a nucleic acid sequence derivatives . The term “ amino acid ” includes both D -amino 20 can , in some cases, alter the underlying template for gene acids and L -amino acids . expression . The terms " derivative , " " variant, ” and “ fragment, ” when A Cas protein referred to herein can be a type of protein used herein with reference to a polypeptide, refers to a or polypeptide . A Cas protein can refer to a nuclease . A Cas polypeptide related to a wild type polypeptide , for example protein can refer to an endoribonuclease . A Cas protein can either by amino acid sequence , structure ( e . g . , secondary 25 refer to any modified ( e . g . , shortened , mutated , lengthened ) and /or tertiary ), activity (e .g . , enzymatic activity ) and /or polypeptide sequence or homologue of the Cas protein . A function . Derivatives , variants and fragments of a polypep - Cas protein can be codon optimized . A Cas protein can be a tide can comprise one or more amino acid variations ( e . g ., codon - optimized homologue of a Cas protein . A Cas protein mutations, insertions , and deletions ), truncations, modifica - can be enzymatically inactive , partially active , constitutively tions , or combinations thereof compared to a wild type 30 active , fully active , inducible active and / or more active , ( e . g . polypeptide . more than the wild type homologue of the protein or The term “ percent ( % ) identity ," as used herein , refers to polypeptide .) . A Cas protein can be Cas9 . A Cas protein can the percentage of amino acid ( or nucleic acid ) residues of a be Cpfi . A Cas protein can be C2c2 . A Cas protein ( e . g . , candidate sequence that are identical to the amino acid (or variant, mutated , enzymatically inactive and / or condition nucleic acid ) residues of a reference sequence after aligning 35 ally enzymatically inactive site - directed polypeptide ) can the sequences and introducing gaps, if necessary , to achieve bind to a target nucleic acid . The Cas protein ( e . g ., variant, the maximum percent identity ( i. e . , gaps can be introduced mutated , enzymatically inactive and / or conditionally enzy in one or both of the candidate and reference sequences for m atically inactive endoribonuclease ) can bind to a target optimal alignment and non -homologous sequences can be RNA or DNA . disregarded for comparison purposes ). Alignment, for pur- 40 The term " crRNA , ” as used herein , can generally refer to poses of determining percent identity , can be achieved in a nucleic acid with at least about 5 % , 10 % , 20 % , 30 % , 40 % , various ways that are within the skill in the art , for instance , 50 % , 60 % , 70 % , 80 % , 90 % , 100 % sequence identity and /or using publicly available computer software such as BLAST, sequence similarity to a wild type exemplary crRNA (e . g ., a ALIGN , or Megalign (DNASTAR ) software . Percent iden - crRNA from S . pyogenes ). crRNA can generally refer to a tity of two sequences can be calculated by aligning a test 45 nucleic acid with at most about 5 % , 10 % , 20 % , 30 % , 40 % , sequence with a comparison sequence using BLAST, deter- 50 % , 60 % , 70 % , 80 % , 90 % , 100 % sequence identity and /or mining the number of amino acids or nucleotides in the sequence similarity to a wild type exemplary crRNA ( e . g . , a aligned test sequence that are identical to amino acids or crRNA from S . pyogenes ) . crRNA can refer to a modified nucleotides in the same position of the comparison form of a crRNA that can comprise a nucleotide change such sequence , and dividing the number of identical amino acids 50 as a deletion , insertion , or substitution , variant , , or or nucleotides by the number of amino acids or nucleotides chimera . A crRNA can be a nucleic acid having at least about in the comparison sequence . 60 % identical to a wild type exemplary crRNA ( e . g ., a The term “ gene modulating polypeptide ” or “ GMP, ” as crRNA from S. pyogenes ) sequence over a stretch of at least used herein , refers to a polypeptide comprising at least an 6 contiguous nucleotides. For example, a crRNA sequence actuator moiety capable of regulating expression or activity 55 can be at least about 60 % identical, at least about 65 % of a gene and / or editing a nucleic acid sequence . A GMP can identical, at least about 70 % identical, at least about 75 % comprise additional peptide sequences which are not identical, at least about 80 % identical, at least about 85 % involved in modulating gene expression , for example cleav - identical, at least about 90 % identical, at least about 95 % age recognition sites, linker sequences , targeting sequences , identical , at least about 98 % identical, at least about 99 % etc . 60 identical , or 100 % identical , to a wild type exemplary The terms " actuator moiety, " " actuator domain ," and crRNA sequence ( e . g ., a crRNA from S . pyogenes ) over a " gene modulating domain , " as used herein , refers to a stretch of at least 6 contiguous nucleotides moiety which can regulate expression or activity of a gene T he term “ tracrRNA , ” as used herein , can generally refer and / or edit a nucleic acid sequence , whether exogenous or to a nucleic acid with at least about 5 % , 10 % , 20 % , 30 % , endogenous. An actuator moiety can regulate expression of 65 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 100 % sequence identity a gene at the transcription level and / or the translation level and /or sequence similarity to a wild type exemplary tracr An actuator moiety can regulate gene expression at the RNA sequence ( e . g . , a tracrRNA from S . pyogenes ) . tracr US 9 ,856 , 497 B2 21 RNA can refer to a nucleic acid with at most about 5 % , 10 % , localization signal (NLS ) ; outside of a nucleus of a cell , for 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 100 % example to the cytoplasm , utilizing a nuclear export signal sequence identity and /or sequence similarity to a wild type (NES ) ; mitochondria utilizing a mitochondrial targeting exemplary tracrRNA sequence ( e . g ., a tracrRNA from S . signal; the endoplasmic reticulum ( ER ) utilizing an ER pyogenes ) . tracrRNA can refer to a modified form of a 5 retention signal; a peroxisome utilizing a peroxisomal tar tracrRNA that can comprise a nucleotide change such as a geting signal; plasmamembrane utilizing a membrane local deletion , insertion , or substitution , variant, mutation , or ization signal; or combinations thereof. chimera . A tracrRNA can refer to a nucleic acid that can be As used herein , “ fusion ” can refer to a protein and /or at least about 60 % identical to a wild type exemplary nucleic acid comprising one or more non - native sequences tracrRNA ( e . g . , a tracrRNA from S . pyogenes ) sequence 10 ( e . g . , moieties ) . A fusion can comprise one or more of the over a stretch of at least 6 contiguous nucleotides . For same non - native sequences . A fusion can comprise one or example , a tracrRNA sequence can be at least about 60 % more of different non - native sequences . A fusion can be a identical, at least about 65 % identical , at least about 70 % chimera . A fusion can comprise a nucleic acid affinity tag . A identical, at least about 75 % identical, at least about 80 % fusion can comprise a barcode . A fusion can comprise a identical , at least about 85 % identical, at least about 90 % 15 peptide affinity tag . A fusion can provide for subcellular identical, at least about 95 % identical, at least about 98 % localization of the site - directed polypeptide ( e .g . , a nuclear identical, at least about 99 % identical, or 100 % identical , to localization signal (NLS ) for targeting to the nucleus, a a wild type exemplary tracrRNA ( e . g . , a tracrRNA from S . mitochondrial localization signal for targeting to the mito pyogenes ) sequence over a stretch of at least 6 contiguous chondria , a chloroplast localization signal for targeting to a nucleotides . 20 chloroplast , an endoplasmic reticulum ( ER ) retention signal , As used herein , a guide nucleic acid can refer to a nucleic and the like ) . A fusion can provide a non -native sequence acid that can hybridize to another nucleic acid . A guide ( e . g ., affinity tag ) that can be used to track or purify . A fusion nucleic acid can be RNA. A guide nucleic acid can be DNA . can be a small molecule such as biotin or a dye such as alexa The guide nucleic acid can be programmed to bind to a fluor dyes , Cyanine3 dye , Cyanine5 dye . sequence of nucleic acid site -specifically . The nucleic acid to 25 A fusion can refer to any protein with a functional effect . be targeted , or the target nucleic acid , can comprise nucleo - For example, a fusion protein can comprise methyltrans tides. The guide nucleic acid can comprise nucleotides. A ferase activity , demethylase activity , dismutase activity , portion of the target nucleic acid can be complementary to alkylation activity , depurination activity , oxidation activity , a portion of the guide nucleic acid . The strand of a double - pyrimidine dimer forming activity , integrase activity , trans stranded target polynucleotide that is complementary to and 30 posase activity , recombinase activity , polymerase activity , hybridizes with the guide nucleic acid can be called the activity , activity, photolyase activity or gly complementary strand . The strand of the double - stranded cosylase activity , acetyltransferase activity , deacetylase target polynucleotide that is complementary to the comple - activity , activity , phosphatase activity , ubiquitin mentary strand , and therefore may not be complementary to ligase activity , deubiquitinating activity , adenylation activ the guide nucleic acid can be called noncomplementary 35 ity , deadenylation activity , SUMOylating activity , deSU strand . A guide nucleic acid can comprise a polynucleotide MOylating activity , ribosylation activity , deribosylation chain and can be called a “ single guide nucleic acid ” . A activity , myristoylation activity , remodelling activity , pro guide nucleic acid can comprise two polynucleotide chains tease activity , oxidoreductase activity , activity , and can be called a " double guide nucleic acid ” . If not activity , activity , activity , syn otherwise specified , the term “ guide nucleic acid ” can be 40 thase activity , synthetase activity , or demyristoylation activ inclusive , referring to both single guide nucleic acids and ity . An effector protein can modify a genomic locus. A fusion double guide nucleic acids . protein can be a fusion in a Cas protein . An fusion protein A guide nucleic acid can comprise a segment that can be can be a non -native sequence in a Cas protein . referred to as a “ nucleic acid - targeting segment” or a As used herein , “ non -native ” can refer to a nucleic acid or “ nucleic acid - targeting sequence . ” A nucleic acid - targeting 45 polypeptide sequence that is not found in a native nucleic nucleic acid can comprise a segment that can be referred to acid or protein . Non - native can refer to affinity tags . Non as a “ protein binding segment" or " protein binding native can refer to fusions. Non -native can refer to a sequence ” or “ Cas protein binding segment ” . naturally occurring nucleic acid or polypeptide sequence The term “ cleavage recognition site , " as used herein , with that comprises mutations , insertions and / or deletions . A reference to peptides , refers to a site of a peptide at which 50 non - native sequence may exhibit and / or encode for an a chemical bond , such as a peptide bond or disulfide bond , activity (e . g ., enzymatic activity , methyltransferase activity , can be cleaved . Cleavage can be achieved by various meth - acetyltransferase activity , kinase activity , ubiquitinating ods. Cleavage of peptide bonds can be facilitated , for activity , etc . ) that can also be exhibited by the nucleic acid example , by an enzyme such as a protease or by protein and / or polypeptide sequence to which the non -native splicing ( e . g . , inteins) . Cleavage of a disulfide bond can be 55 sequence is fused . A non -native nucleic acid or polypeptide facilitated , for example, by an enzyme such as an oxi- sequence may be linked to a naturally - occurring nucleic acid doreductase . or polypeptide sequence (or a variant thereof) by genetic The term “ targeting sequence , " as used herein , refers to a engineering to generate a chimeric nucleic acid and /or nucleotide sequence and the corresponding amino acid polypeptide sequence encoding a chimeric nucleic acid sequence which encodes a targeting polypeptide which 60 and / or polypeptide . mediates the localization ( or retention ) of a protein to a As used herein , “ treating ” or “ treatment” refers to any one sub - cellular location , e . g ., plasma membrane or membrane of the following : ameliorating one or more symptoms of of a given organelle , nucleus, cytosol, mitochondria, endo - disease , e . g . , cancer, preventing the manifestation of such plasmic reticulum ( ER ), Golgi, chloroplast , apoplast , per - symptoms before they occur, slowing down or completely oxisome or other organelle . For example , a targeting 65 preventing the progression of the disease ( as may be evident sequence can direct a protein (e .g . , a receptor polypeptide or by longer periods between reoccurrence episodes , slowing an adaptor polypeptide ) to a nucleus utilizing a nuclear down or prevention of the deterioration of symptoms, etc . ); US 9 ,856 , 497 B2 23 24 enhancing the onset of a remission period ; slowing down the In some embodiments , ( i) the chimeric receptor polypep irreversible damage caused in the progressive -chronic stage tide is modified in response to antigen binding , (ii ) the of the disease (both in the primary and secondary stages ) ; cleavage recognition site is cleaved by a cleavage moiety in delaying the onset of said progressive stage, or any combi response to modification of the chimeric receptor polypep nation thereof. 5 tide, (iii ) the actuator moiety complexes with a target As used herein , “ administer, " " administering, " " adminis polynucleotide after being cleaved from the chimeric recep tration ," and derivatives thereof refer to the methods that tor polypeptide at the cleavage recognition site , and ( iv ) the may be used to enable delivery of agents or compositions to chimeric receptor polypeptide does not comprise SEQ ID the desired site of biological action . These methods include , NO : 39 . but are not limited to parenteral administration ( e . g . , intra - 10 venous, subcutaneous , intraperitoneal , intramuscular, intra vascular , intrathecal, intranasal, intravitreal, infusion and SEQ ID NO : 39 local injection ) , transmucosal injection , oral administration , ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDG administration as a suppository, and topical administration . Administration is by any route , including parenteral. Paren - 15 GDCSLNFNDPWKNCTOSLQCWKYFSDGHCDSQCNSAGCLFDGFDCOLTEG teral administration includes , e . g ., intravenous , intramuscu - QCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVL lar, intra - arteriole , intradermal, subcutaneous, intraperito neal, intraventricular, and intracranial . Other modes of VLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQOMIFPYYGHEEEL delivery include, but are not limited to , the use of liposomal RKHPIKRSTVGWATSSLLPGTSGGRORRELDPMDIRGSIVYLEIDNRQCV formulations, intravenous infusion , transplantation , etc . One 20 skilled in the art will know of additional methods for QSSSQCFOSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHL administering a therapeutically effective amount of a com position of the present disclosure for preventing or relieving MYVAAAAFVLLFFVGCGVLLSRKRRR one or more symptoms associated with a disease . A chimeric receptor polypeptide of a subject system can Disclosed herein are systems, methods, and compositions 25 comprise an endogenous receptor, or any derivative , variant for regulating expression of a target polynucleotide in a cell. or fragment thereof. A chimeric receptor polypeptide can In an aspect , the present disclosure provides a system for b ind specifically to at least one antigen ( e . g . , at least one regulating expression of a target polynucleotide in a cell . An ligand ) , for example via an antigen interacting domain ( also exemplary system comprises ( a ) a chimeric receptor poly - referred to herein as an “ extracellular sensor domain ” ) . A peptide that is modified upon binding an antigen , wherein 30 chimeric receptor polypeptide can , in response to ligand receptor modification comprises a conformational change or b inding , undergo a modification such as a conformational chemical modification , ( b ) a chimeric adaptor polypeptide change and /or chemical modification . Such modification ( s ) that binds the receptor in response to the receptor modifi - can recruit to the receptor binding partners ( e . g . , partners cation , ( c ) a gene modulating polypeptide (GMP ) compris such as proteins ) including , but not limited to , signaling ing an actuator moiety linked to a cleavage recognition site , 35 proteins involved in signaling events and various cellular wherein upon cleavage of the cleavage recognition site , the processes. Signaling proteins, for example , can be involved actuator moiety is activated to complex with a target poly - in regulating ( e . g . , activating and /or de - activating ) a cellular nucleotide , and ( d ) a cleavage moiety that cleaves the response such as programmed changes in gene expression cleavage recognition site when in proximity to the cleavage via translational regulation ; transcriptional regulation ; and recognition site . The chimeric receptor polypeptide , chime - 40 epigenetic modification including the regulation of methyl ric adaptor polypeptide, gene modulating polypeptide a tion , acetylation , phosphorylation , ubiquitylation , sumoy (GMP ) , and cleavage moiety of a subject system can be lation , ribosylation , and citrullination . Conformational arranged in a variety of configurations . Exemplary , non - changes of a chimeric receptor polypeptide can expose one limiting configurations are described herein . In some or more regions of the receptor which was previously not embodiments , the GMP forms a portion of an intracellular 45 exposed , and the exposed region can recruit and / or bind region of the chimeric receptor polypeptide , and the cleav - signaling protein ( s ). Chemical modifications on a receptor, age moiety forms a portion of the chimeric adaptor poly - for example phosphorylation and / or dephosphorylation peptide . In some embodiments , the GMP forms a portion of ( e . g ., at tyrosine, , , and / or any other suitable the chimeric adaptor polypeptide , and the cleavage moiety amino acid residue ), can also recruit signaling proteins forms a portion of an intracellular region of the chimeric 50 involved in regulating intracellular processes . Signaling receptor polypeptide . In some embodiments , the cleavage proteins can bind directly to a receptor or indirectly to a moiety is complexed with a second adaptor polypeptide that receptor, for example as part of a larger complex . binds the chimeric receptor polypeptide in response to the In some embodiments , the chimeric receptor polypeptide receptor modification , and the GMP forms a portion of the is a transmembrane receptor. An exemplary transmembrane chimeric adaptor polypeptide . 55 receptor is shown in FIG . 2 . A transmembrane receptor can In an exemplary configuration , the GMP forms a portion be embedded in a and have at least an of an intracellular region of the chimeric receptor polypep - extracellular region 201 , a region spanning a membrane 202 tide , and the cleavage moiety forms a portion of the chimeric such as a plasmamembrane , and an intracellular region 203 . adaptor polypeptide. A chimeric receptor polypeptide of an The antigen interacting domain can form a portion of the exemplary configuration can comprise ( a ) an antigen inter - 60 extracellular region , and the GMP can form a portion of the acting domain , and ( b ) a gene modulating polypeptide intracellular region . Membrane receptors can detect at least (GMP ) comprising an actuator moiety linked to a cleavage one signal, such as a small molecule , ion , or protein , from recognition site . FIG . 1 shows an exemplary chimeric recep the surrounding environment ( e . g . , extracellular and/ or tor polypeptide . The receptor comprises an antigen interact - intracellular environment) and can initiate a cellular ing domain 101 and a gene modulating polypeptide (GMP ) 65 response via at least one signaling cascade involving addi 102 . The GMP 102 can comprise an actuator moiety 102a tional proteins and signaling molecules . Some receptors can linked to a cleavage recognition site 102b . translocate from one region of a cell to another, for example US 9 ,856 , 497 B2 25 26 from the plasma membrane or cytoplasm to the nucleus and thereof. A chimeric receptor polypeptide comprising a vice versa . Such translocation can be conditional upon GPCR , or any derivative , variant or fragment thereof, can ligand binding to the receptor. Examples of membrane recruit a binding partner. In some embodiments , ligand receptors include , but are not limited to , Notch receptors ; binding to a chimeric receptor comprising a GPCR , or any G - protein coupled receptors (GPCRs ) ; integrin receptors ; 5 derivative , variant or fragment thereof, results in a confor cadherin receptors ; catalytic receptors including receptors mational change , chemical modification , or combination possessing enzymatic activity and receptors which , rather thereof , which recruits a binding partner to the receptor . than possessing intrinsic enzymatic activity , act by stimu- In some embodiments , a chimeric receptor polypeptide lating non - covalently associated ( e . g ., ) ; comprises a GPCR , or any derivative , variant or fragment death receptors such as members of the tumor necrosis 10 thereof, selected from Class A Orphans ; Class B Orphans ; factor receptor ( TNFR ) superfamily ; and immune receptors . Class C Orphans ; taste receptors , type 1 ; taste receptors , In some embodiments , a chimeric receptor polypeptide type 2 ; 5 -hydroxytryptamine receptors ; acetylcholine recep comprises a Notch receptor , or any derivative , variant or tors (muscarinic ) ; adenosine receptors ; adhesion class fragment thereof. Notch receptors are transmembrane pro GPCRs ; adrenoceptors ; receptors ; recep teins that mediate cell - cell contact signaling and play a 15 tor ; bile acid receptor ; receptors ; central role in development and other aspects of cell - to - cell receptors ; receptors ; -sensing receptors ; communication , e . g . communication between two contact - cannabinoid receptors ; chemerin receptor, chemokine recep ing cells (receiver cell and sending cell ) . Notch receptors tors ; receptors ; class GPCRs ( e . g . , expressed in a receiver cell recognize their ligands ( the delta Wnt receptors) ; complement peptide receptors; corticotro family of proteins ) , expressed on a sending cell. The engage - 20 pin -releasing factor receptors ; receptors ; endothe ment of notch and delta on these contacting cells leads to lin receptors ; G protein -coupled receptor ; formyl two- step of the notch receptor that ultimately p eptide receptors; free fatty acid receptors ; GABAB causes the release of the intracellular portion of the receptor receptors ; receptors ; receptor ; from the membrane into the cytoplasm . receptor family ; glycoprotein hormone receptors ; gonado In some embodiments , a chimeric receptor polypeptide 25 trophin - releasing hormone receptors ; GPR18 , GPR55 and comprises at least an extracellular region ( e .g . , ligand bind GPR119 ; histamine receptors ; hydroxycarboxylic acid ing domain ) of a Notch receptor , or any derivative, variant receptors; receptor ; leukotriene receptors ; lyso or fragment thereof. In some embodiments , a chimeric phospholipid (LPA ) receptors ; lysophospholipid (S1P ) receptor polypeptide comprises at least a membrane span - receptors ; - concentrating hormone receptors ; mel ning region of a Notch , or any derivative , variant or frag - 30 anocortin receptors ; receptors ; metabotropic glu ment thereof . In some embodiments , a chimeric receptor tamate receptors; receptor ; receptors ; polypeptide comprises at least an intracellular region ( e . g ., FF /neuropeptide AF receptors ; cytoplasmic domain ) of a Notch , or any derivative , variant receptor; neuropeptide W / receptors ; neuro or fragment thereof. A chimeric receptor polypeptide com peptide Y receptors; receptors ; opioid receptors ; prising a Notch , or any derivative , variant or fragment 35 receptors ; oxoglutarate receptor ; P2Y receptors ; para thereof, can recruit a binding partner. In some embodiments , hormone receptors; - activating factor recep ligand binding to a chimeric receptor comprising a Notch , or tor; prokineticin receptors ; - releasing peptide any derivative, variant or fragment thereof, results in a receptor; receptors ; proteinase - activated recep conformational change , chemical modification , or combina - tors ; QRFP receptor ; family peptide receptors ; tion thereof, which recruits a binding partner to the receptor. 40 tostatin receptors ; succinate receptor; tachykinin receptors ; In some embodiments , a chimeric receptor polypeptide thyrotropin - releasing hormone receptors ; trace amine recep comprises a Notch , or any derivative , variant or fragment tor ; urotensin receptor ; and receptors ; thereof, selected from Notch1, Notch2 , Notch3 , and Notch4 VIP and PACAP receptors . or any homolog thereof. In some embodiments , a chimeric receptor polypeptide In some embodiments , a chimeric receptor polypeptide 45 comprises a GPCR selected from the group consisting of: comprises a G - protein coupled receptor (GPCR ), or any 5 -hydroxytryptamine (serotonin ) receptor 1A (HTR1A ) , derivative , variant or fragment thereof. GPCRs are generally 5 - hydroxytryptamine ( serotonin ) receptor 1B (HTR1B ) , characterized by seven membrane - spanning a helices and 5 - hydroxytryptamine serotonin ) receptor 1D (HTRID ) , can be arranged in a tertiary structure resembling a barrel, 5 - hydroxytryptamine ( serotonin ) receptor 1E (HTRIE ) , with the seven transmembrane helices forming a cavity 50 5 - hydroxytryptamine ( serotonin ) receptor 1F (HTR1F ) , within the plasma membrane that serves as a ligand -binding 5 - hydroxytryptamine ( serotonin ) receptor 2A (HTR2A ) , domain . Ligands can also bind elsewhere to a GPCR , for 5 -hydroxytryptamine (serotonin ) receptor 2B (HTR2B ) , example to the extracellular loops and / or the N -terminal tail. 5 - hydroxytryptamine ( serotonin ) receptor 2C (HTR2C ) , Ligand binding can activate an associated G protein , which 5 -hydroxytryptamine ( serotonin ) receptor 4 (HTR4 ) , 5 -hy then functions in various signaling pathways . To de -activate 55 droxytryptamine ( serotonin ) receptor 5A (HTR5A ) , 5 -hy this signaling , a GPCR can first be chemically modified by droxytryptamine ( serotonin ) receptor 5B (HTR5BP ), 5 - hy phosphorylation . Phosphorylation can then recruit co -adap droxytryptamine ( serotonin receptor 6 (HTR6 ) , tor proteins ( e . g . , arrestin proteins ) for additional signaling . 5 - hydroxytryptamine serotonin ) receptor 7 , adenylate In some embodiments, a chimeric receptor polypeptide cyclase - coupled (HTR7 ) , receptor, muscarinic 1 comprises at least an extracellular region (e . g ., ligand bind - 60 (CHRM1 ) , cholinergic receptor, muscarinic 2 (CHRM2 ) , ing domain ) of a GPCR , or any derivative , variant or cholinergic receptor, muscarinic 3 (CHRM3 ) , cholinergic fragment thereof. In some embodiments , a chimeric receptor receptor, muscarinic 4 (CHRM4 ) , cholinergic receptor, mus polypeptide comprises at least a membrane spanning region carinic 5 (CHRM5 ) , adenosine Al receptor (ADORA1 ) , of a GPCR , or any derivative , variant or fragment thereof. In (ADORA2A ), adenosine A2b recep some embodiments, a chimeric receptor polypeptide com - 65 tor (ADORA2B ) , (ADORA3 ) , adhe prises at least an intracellular region ( e . g ., cytoplasmic sion G protein - coupled receptor A1 ( ADGRA1) , adhesion G domain ) of a GPCR , or any derivative , variant or fragment protein - coupled receptor A2 ( ADGRA2 ) , adhesion G pro US 9 ,856 ,497 B2 27 28 tein - coupled receptor A3 (ADGRA3 ), adhesion G protein receptor (CCKBR ), G protein -coupled receptor 1 (GPR1 ) , coupled receptor B1 ( ADGRB1) , adhesion G protein bombesin like receptor 3 (BRS3 ), G protein - coupled recep coupled receptor B2 ( ADGRB2) , adhesion G protein tor 3 (GPR3 ) , G protein - coupled receptor 4 (GPR4 ), G coupled receptor B3 (ADGRB3 ), cadherin EGF LAG seven protein -coupled receptor 6 (GPR6 ) , G protein - coupled pass G - type receptor 1 (CELSR1 ) , cadherin EGF LAG 5 receptor 12 (GPR12 ) , G protein - coupled receptor 15 seven -pass G - type receptor 2 (CELSR2 ) , cadherin EGF (GPR15 ), G protein - coupled receptor 17 (GPR17 ), G pro LAG seven - pass G - type receptor 3 (CELSR3 ) , adhesion G tein - coupled receptor 18 (GPR18 ) , G protein - coupled recep protein -coupled receptor D1 ( ADGRD1) , adhesion G pro tor 19 (GPR19 ) , G protein - coupled receptor 20 (GPR20 ) , G tein -coupled receptor D2 (ADGRD2 ) , adhesion G protein protein - coupled receptor 21 (GPR21 ), G protein -coupled coupled receptor E1 ( ADGRE1) , adhesion G protein - 10 receptor 22 (GPR22 ) , G protein -coupled receptor 25 coupled receptor E2 ( ADGRE2 ) , adhesion G protein (GPR25 ) , G protein - coupled receptor 26 (GPR26 ) , G pro coupled receptor E3 ( ADGRE3 ) , adhesion G protein tein - coupled receptor 27 (GPR27 ) , G protein - coupled recep coupled receptor E4 (ADGRE4P ), adhesion G protein tor 31 (GPR31 ) , G protein - coupled receptor 32 (GPR32 ) , G coupled receptor E5 ( ADGRE5 ) , adhesion G protein protein -coupled receptor 33 ( gene / ) (GPR33 ) , G coupled receptor F1 ( ADGRF1) , adhesion G protein - 15 protein -coupled receptor 34 (GPR34 ) , G protein -coupled coupled receptor F2 (ADGRF2 ) , adhesion G protein receptor 35 (GPR35 ) , G protein -coupled receptor 37 ( en coupled receptor F3 ( ADGRF3 ) , adhesion G protein dothelin receptor type B - like ) (GPR37 ) , G protein -coupled coupled receptor F4 ( ADGRF4) , adhesion G protein receptor 37 like 1 (GPR37L1 ) , G protein - coupled receptor coupled receptor F5 ( ADGRF5 ) , adhesion G protein - 39 (GPR39 ) , G protein - coupled receptor 42 ( gene /pseudo coupled receptor G1 (ADGRG1 ) , adhesion G protein - 20 gene ) (GPR42 ), G protein - coupled receptor 45 (GPR45 ) , G coupled receptor G2 (ADGRG2 ) , adhesion G protein protein - coupled receptor 50 (GPR50 ) , G protein - coupled coupled receptor G3 ( ADGRG3 ), adhesion G protein receptor 52 (GPR52 ) , G protein - coupled receptor 55 coupled receptor G4 ( ADGRG4 ), adhesion G protein (GPR55 ) , G protein -coupled receptor 61 (GPR61 ) , G pro coupled receptor G5 ( ADGRG5 ) , adhesion G protein tein -coupled receptor 62 (GPR62 ) , G protein - coupled recep coupled receptor G6 ( ADGRG6 ) , adhesion G protein - 25 tor 63 (GPR63 ) , G protein -coupled receptor 65 (GPR65 ) , G coupled receptor G7 ( ADGRG7) , adhesion G protein - protein - coupled receptor 68 (GPR68 ) , G protein - coupled coupled receptor ( ADGRL1) , adhesion G protein receptor 75 (GPR75 ), G protein -coupled receptor 78 coupled receptor L2 (ADGRL2 ), adhesion G protein (GPR78 ), G protein -coupled receptor 79 (GPR79 ), G pro coupled receptor L3 (ADGRL3 ) , adhesion G protein - tein - coupled receptor 82 (GPR82 ) , G protein - coupled recep coupled receptor L4 ( ADGRL4) , adhesion G protein - 30 tor 83 (GPR83 ) , G protein - coupled receptor 84 (GPR84 ), G coupled receptor V1 (ADGRV1 ) , adrenoceptor alpha 1A protein - coupled receptor 85 (GPR85 ) , G protein - coupled ( ADRA1A ) , adrenoceptor alpha 1B (ADRA1B ) , adrenocep - receptor 87 (GPR87 ) , G protein - coupled receptor 88 tor alpha 1D ( ADRA1D ) , adrenoceptor alpha 2A (GPR88 ), G protein -coupled receptor 101 (GPR101 ), G ( ADRA2A ) , adrenoceptor alpha 2B ( ADRA2B ) , adrenocep protein -coupled receptor 119 (GPR119 ) , G protein - coupled tor alpha 2C ( ADRA2C ) , adrenoceptor beta 1 ( ADRB1) , 35 receptor 132 (GPR132 ), G protein - coupled receptor 135 adrenoceptor beta 2 ( ADRB2 ) , adrenoceptor beta 3 (GPR135 ) , G protein - coupled receptor 139 (GPR139 ) , G ( ADRB3 ) , angiotensin II receptor type 1 (AGTR1 ) , angio protein - coupled receptor 141 (GPR141 ) , G protein -coupled tensin II receptor type 2 (AGTR2 ) , receptor 142 (GPR142 ), G protein - coupled receptor 146 ( APLNR ) , G protein - coupled bile acid receptor 1 (GP (GPR146 ) , G protein - coupled receptor 148 (GPR148 ) , G BAR1) , receptor (NMBR ) , releasing 40 protein -coupled receptor 149 (GPR149 ), G protein - coupled peptide receptor (GRPR ) , bombesin like receptor 3 (BRS3 ) , receptor 150 (GPR150 ), G protein - coupled receptor 151 B1 ( BDKRB1) , (GPR151 ) , G protein - coupled receptor 152 (GPR152 ) , G (BDKRB2 ) , (CALCR ), calcitonin recep - protein -coupled receptor 153 (GPR153 ) , G protein - coupled tor like receptor (CALCRL ) , calcium sensing receptor receptor 160 (GPR160 ), G protein - coupled receptor 161 ( CASR ), G protein -coupled receptor, class C (GPRC6A ), 45 (GPR161 ) , G protein - coupled receptor 162 (GPR162 ), G 1 (brain ) (CNR1 ) , cannabinoid recep - protein -coupled receptor 171 (GPR171 ) , G protein - coupled tor 2 (CNR2 ), chemerin chemokine - like receptor 1 (CM - receptor 173 (GPR173 ) , G protein - coupled receptor 174 KLR1) , chemokine (C - C motif ) receptor 1 (CCR1 ), (GPR174 ), G protein - coupled receptor 176 (GPR176 ), G chemokine ( C - C motif ) receptor 2 (CCR2 ), chemokine ( C - C protein - coupled receptor 182 (GPR182 ) , G protein - coupled motif ) receptor 3 ( CCR3) , chemokine (C -C motif ) receptor 50 receptor 183 (GPR183 ), - rich repeat containing G 4 (CCR4 ) , chemokine ( C - C motif ) receptor 5 ( gene / pseudo - protein -coupled receptor 4 (LGR4 ) , leucine - rich repeat con gene ) (CCRS ) , chemokine ( C - C motif ) receptor 6 (CCR6 ) , taining G protein -coupled receptor 5 (LGR5 ), leucine -rich chemokine ( C - C motif ) receptor 7 (CCR7 ) , chemokine ( C - C repeat containing G protein - coupled receptor 6 (LGR6 ) , motif ) receptor 8 (CCR8 ) , chemokine ( C - C motif ) receptor MASI proto -oncogene (MAS 1 ), MASI proto -oncogene like 9 ( CCRI) , chemokine ( C - C motif ) receptor 10 (CCR10 ), 55 (MASIL ) , MAS related GPR family member D (MRG chemokine ( C - X - C motif ) receptor 1 ( CXCR1) , chemokine PRD ), MAS related GPR family member E (MRGPRE ) , ( C - X - C motif ) receptor 2 (CXCR2 ) , chemokine ( C - X - C MAS related GPR family member F (MRGPRF ) , MAS motif ) receptor 3 (CXCR3 ) , chemokine ( C - X -C motif ) related GPR family member G (MRGPRG ), MAS related receptor 4 (CXCR4 ) , chemokine ( C - X - C motif ) receptor 5 GPR family member X1 (MRGPRX1 ), MAS related GPR (CXCR5 ), chemokine ( C - X - C motif ) receptor 6 (CXCR6 ) , 60 family member X2 (MRGPRX2 ) , MAS related GPR family chemokine ( C - X3 - C motif ) receptor 1 (CX3CR1 ) , member X3 (MRGPRX3 ) , MAS related GPR family mem chemokine (C motif ) receptor 1 (XCR1 ) , atypical ber X4 (MRGPRX4 ) , 3 (OPN3 ), opsin 4 (OPN4 ) , 1 (Duffy blood group ) ( ACKR1) , atypi opsin 5 (OPNS ) , P2Y (P2RY8 ) , puriner cal chemokine receptor 2 (ACKR2 ) , atypical chemokine gic receptor P2Y ( P2RY10 ), trace amine associated receptor receptor 3 ( ACKR3 ), atypical chemokine receptor 4 65 2 ( TAAR2) , trace amine associated receptor 3 ( gene /pseudo ( ACKR4 ) , chemokine ( C - C motif ) receptor - like 2 ( CCRL2 ), gene ) ( TAAR3 ) , trace amine associated receptor 4 cholecystokinin A receptor ( CCKAR ) , cholecystokinin B ( TAAR4P ) , trace amine associated receptor 5 ( TAAR5) , US 9 ,856 ,497 B2 29 30 trace amine associated receptor 6 ( TAARO ) , trace amine receptor (adrenocorticotropic hormone ) (MC2R ), melano associated receptor 8 ( TAAR8 ) , trace amine associated cortin 3 receptor (MC3R ) , 4 receptor (MC4R ) , receptor 9 ( gene / pseudogene ) ( TAAR9) , G protein - coupled (MC5R ), 1A receptor 156 (GPR156 ) , G protein - coupled receptor 158 (MTNR1A ) , (MTNR1B ), glutamate (GPR158 ) , G protein -coupled receptor 179 (GPR179 ), G 5 receptor, metabotropic 1 (GRM1 ) , , protein - coupled receptor, class C (GPRC5A ) , G protein - metabotropic 2 (GRM2 ) , glutamate receptor, metabotropic 3 coupled receptor, class C (GPRC5B ), G protein - coupled (GRM3 ) , glutamate receptor , metabotropic 4 (GRM4 ), glu receptor, class C (GPRC5C ), G protein - coupled receptor , tamate receptor, metabotropic 5 (GRM5 ) , glutamate recep class C (GPRC5D ) , frizzled class receptor 1 (FZD1 ) , tor, metabotropic 6 (GRM6 ) , glutamate receptor, metabo frizzled class receptor 2 (FZD2 ), frizzled class receptor 3 10 tropic 7 (GRM7 ) , glutamate receptor, metabotropic 8 (FZD3 ) , frizzled class receptor 4 ( FZD4 ) , frizzled class GRM8( ) , (MLNR ) , receptor 5 (FZD5 ) , frizzled class receptor 6 ( FZD6 ) , frizzled 1 (NMUR1 ) , (NMUR2 ) , neuro class receptor 7 (FZD7 ) , frizzled class receptor 8 (FZD8 ) , peptide FF receptor 1 (NPFFR1 ) , neuropeptide FF receptor frizzled class receptor 9 (FZD9 ) , frizzled class receptor 10 2 (NPFFR2 ), 1 ( NPSR1) , neuro ( FZD10 ), , frizzled class receptor (SMO ), 15 peptides B / W receptor 1 (NPBWR1 ) , B / W complement component 3a receptor 1 (C3AR1 ) , comple - receptor 2 (NPBWR2 ) , receptor Y1 ment component 5a receptor 1 (C5AR1 ) , complement com (NPY1R ) , Y2 (NPY2R ), neuropep ponent 5a receptor 2 (C5AR2 ) , corticotropin releasing hor tide Y receptor Y4 (NPY4R ), neuropeptide Y receptor Y5 mone receptor 1 (CRHR1 ) , corticotropin releasing hormone (NPY5R ), neuropeptide Y receptor Y6 ( pseudogene ) receptor 2 (CRHR2 ), D1 (DRD1 ), dop - 20 (NPY6R ), 1 (high affinity ) (NTSR1 ) , amine receptor D2 (DRD2 ) , (DRD3 ) , (NTSR2 ) , , delta 1 (DRD4 ) , (OPRD1 ) , opioid receptor, kappa 1 (OPRK1 ) , opioid recep ( DRD5) , receptor type A (EDNRA ), endothelin tor, mu 1 (OPRM1 ), opiate receptor- like 1 (OPRL1 ) , hypo receptor type B ( EDNRB ), 1 cretin (orexin ) receptor 1 (HCRTR1 ) , hypocretin ( orexin ) ( FPR1) , formyl peptide receptor 2 ( FPR2 ) , formyl peptide 25 receptor 2 (HCRTR2 ), G protein - coupled receptor 107 receptor 3 (FPR3 ), 1 (FFAR1 ) , free (GPR107 ) , G protein -coupled receptor 137 (GPR137 ), fatty acid receptor 2 (FFAR2 ), free fatty acid receptor 3 family 51 subfamily E member 1 ( FFAR3) , free fatty acid receptor 4 (FFAR4 ) , G protein - (OR51E1 ) , transmembrane protein , adipocyte associated 1 coupled receptor 42 ( gene /pseudogene ) (GPR42 ) , gamma - ( TPRA1) , G protein - coupled receptor 143 (GPR143 ) , G aminobutyric acid (GABA ) B receptor, 1 (GABBR1 ) , 30 protein -coupled receptor 157 (GPR157 ) , oxoglutarate ( al gamma- aminobutyric acid (GABA ) B receptor, 2 pha -ketoglutarate ) receptor 1 (OXGR1 ) , purinergic receptor (GABBR2 ), 1 (GALR1 ) , P2Y (P2RY1 ) , purinergic receptor P2Y (P2RY2 ) , pyrimi (GALR2 ) , (GALR3 ) , dinergic receptor P2Y (P2RY4 ) , pyrimidinergic receptor secretagogue receptor (GHSR ) , growth hormone releasing P2Y (P2RY6 ), purinergic receptor P2Y (P2RY11 ) , puriner (GHRHR ), gastric inhibitory polypeptide 35 gic receptor P2Y (P2RY12 ) , purinergic receptor (GIPR ) , glucagon like peptide 1 receptor (GLP1R ), (P2RY13 ), purinergic receptor P2Y (P2RY14 ) , parathyroid glucagon - like peptide 2 receptor (GLP2R ), glucagon recep hormone 1 receptor (PTH1R ) , 2 recep tor (GCGR ) , receptor ( SCTR ) , follicle stimulating tor (PTH2R ) , platelet - activating factor receptor (PTAFR ) , hormone receptor (FSHR ), luteinizing hormone/ choriogo 1 (PROKR1 ) , nadotropin receptor (LHCGR ), thyroid stimulating hormone 40 (PROKR2 ) , prolactin releasing hormone receptor (PRLHR ), receptor ( TSHR ) , gonadotropin releasing hormone receptor D2 receptor (DP ) (PTGDR ), (GNRHR ), gonadotropin releasing hormone receptor 2 receptor 2 (PTGDR2 ) , prostaglandin E receptor 1 ( PT (pseudogene ) (GNRHR2 ), G protein -coupled receptor 18 GER1) , prostaglandin E receptor 2 (PTGER2 ), prostaglan (GPR18 ) , G protein - coupled receptor 55 (GPR55 ) , G pro - din E receptor 3 (PTGER3 ) , prostaglandin E receptor 4 tein - coupled receptor 119 (GPR119 ) , G protein - coupled 45 (PTGER4 ) , (PTGFR ) , prostaglan 1 (GPER1 ) , H1 din 12 ( ) receptor (IP ) (PTGIR ) , thromboxane (HRH1 ) , histamine receptor H2 (HRH2 ) , histamine receptor A2 receptor ( TBXA2R ) , factor II thrombin H3 (HRH3 ) , histamine receptor H4 (HRH4 ), hydroxycar receptor (F2R ), F2R like trypsin receptor 1 ( F2RL1) , coagu boxylic acid receptor 1 (HCAR1 ) , hydroxycarboxylic acid lation factor II like 2 ( F2RL2 ) , F2R like receptor 2 (HCAR2 ) , hydroxycarboxylic acid receptor 3 50 thrombin / trypsin receptor 3 (F2RL3 ) , pyroglutamylated (HCAR3 ) , KISS1 receptor (KISS1R ), recep - RFamide peptide receptor ( QRFPR ) , relaxin / - like tor ( LTB4R ) , 2 ( LTB4R2) , cysteinyl family peptide receptor 1 (RXFP1 ) , relaxin / insulin - like fam 1 (CYSLTR1 ) , cysteinyl leukotriene ily peptide receptor 2 (RXFP2 ) , relaxin /insulin -like family receptor 2 ( CYSLTR2) , oxoeicosanoid (OXE ) receptor 1 peptide receptor 3 (RXFP3 ) , relaxin / insulin - like family pep (OXER1 ), formyl peptide receptor 2 (FPR2 ), lysophospha - 55 tide receptor 4 (RXFP4 ), receptor 1 (SSTR1 ), tidic acid receptor 1 (LPAR1 ) , recep 2 (SSTR2 ), tor 2 (LPAR2 ) , lysophosphatidic acid receptor 3 (LPAR3 ) , (SSTR3 ) , (SSTR4 ) , somatostatin lysophosphatidic acid receptor 4 ( LPAR4 ) , lysophosphatidic receptor 5 (SSTR5 ) , succinate receptor 1 (SUCNR1 ) , tachy acid receptor 5 (LPAR5 ) , lysophosphatidic acid receptor 6 receptor 1 ( TACR1) , 2 ( TACR2 ), (LPAR6 ) , sphingosine - 1 - phosphate receptor 1 (S1PR1 ) , 60 ( TACR3 ) , taste 1 receptor member 1 sphingosine - 1- phosphate receptor 2 ( S1PR2) , sphingosine - ( TAS1R1 ) , taste 1 receptor member 2 ( TAS1R2 ) , taste 1 1 -phosphate receptor 3 (S1PR3 ) , sphingosine - 1 -phosphate receptor member 3 ( TAS1R3) , taste 2 receptor member 1 receptor 4 (S1PR4 ), sphingosine- 1 -phosphate receptor 5 (TAS2R1 ), taste 2 receptor member 3 ( TAS2R3) , taste 2 (SIPR5 ) , melanin concentrating hormone receptor 1 receptor member 4 ( TAS2R4 ) , taste 2 receptor member 5 (MCHR1 ), melanin concentrating hormone receptor 2 65 (TAS2R5 ), taste 2 receptor member 7 ( TAS2R7) , taste 2 (MCHR2 ) , ( alpha receptor member 8 ( TAS2R8 ) , taste 2 receptor member 9 stimulating hormone receptor ) (MC1R ) , melanocortin 2 (TAS2R9 ) , taste 2 receptor member 10 ( TAS2R10 ), taste 2 US 9 ,856 ,497 B2 31 32 receptor member 13 ( TAS2R13) , taste 2 receptor member 14 lysophosphatidylserine, Medium - chain - length fatty acids, ( TAS2R14 ), taste 2 receptormember 16 ( TAS2R16 ) , taste 2 melanin - concentrating hormone , melatonin , methylcar receptor member 19 ( TAS2R19 ), taste 2 receptormember 20 bamyl PAF , Mg2 + , motilin , N -arachidonoylglycine , neuro ( TAS2R20 ) , taste 2 receptor member 30 ( TAS2R30 ) , taste 2 kinin A , neurokinin B , neuromedin B , , neu receptor member 31 ( TAS2R31) , taste 2 receptor member 38 5 romedin S - 33 , neuromedin U - 25 , neuronostatin , ( TAS2R38 ) , taste 2 receptor member 39 ( TAS2R39 ) , taste 2 neuropeptide AF , neuropeptide B - 23 , neuropeptide B - 29 , receptor member 40 ( TAS2R40 ), taste 2 receptor member 41 neuropeptide FF , neuropeptide S , neuropeptide SF, neuro ( TAS2R41 ) , taste 2 receptor member 42 ( TAS2R42 ) , taste 2 peptide W -23 , neuropeptide W -30 , neuropeptide Y , neuro receptor member 43 ( TAS2R43 ) , taste 2 receptormember 45 peptide Y - ( 3 - 36 ) , neurotensin , nociceptin /orphanin FQ , ( TAS2R45 ) , taste 2 receptormember 46 ( TAS2R46 ), taste 2 10 N -oleoylethanolamide , , octopamine , orexin - A , rereceptor member 50 ( TAS2R50 ), taste 2 receptor member 60 orexin - B , Oxysterols , oxytocin , PACAP - 27 , PACAP - 38 , ( TAS2R60 ), thyrotropin - releasing hormone receptor PAF , , peptide YY , PGD2, PGE2 , ( TRHR ) , trace amine associated receptor 1 ( TAAR1) , uro PGF2a , PGI2 , PGJ2 , PHM , phosphatidylserine , PHV , pro tensin 2 receptor (UTS2R ), 1A kineticin - 1 , prokineticin -2 , prokineticin - 2ß , prosaposin , (AVPR1A ) , arginine ( AVPR1B ) , 15 PrRP - 20 , PrRP -31 , PTH , PTHrP , PTHrP - ( 1 - 36 ), QRFP43 , arginine (AVPR2 ) , relaxin , relaxin - 1 , relaxin - 3 , resolvin Di, resolvin El , (OXTR ), adenylate cyclase activating polypeptide 1 (pitu - RFRP - 1 , RFRP -3 , R - spondins , secretin , serine proteases , itary ) receptor type I ( ADCYAP1R1) , vasoactive intestinal sphingosine 1 -phosphate , sphingosylphosphorylcholine , peptide receptor 1 ( VIPR1) , vasoactive intestinal peptide SRIF - 14 , SRIF - 28 , substance P , succinic acid , thrombin , receptor 2 (VIPR2 ), any derivative thereof , any variant 20 , TIP39 , T -kinin , TRH , TSH , tyramine, thereof, and any fragment thereof. UDPUDI - glucose , , 1 , urocortin 2 , A chimeric receptor polypeptide comprising a GPCR , or urocortin 3 , urotensin II - related peptide , urotensin - II, vaso any derivative , variant or fragment thereof, can bind an pressin , VIP , Wnt, Wnt- 1 , Wnt- 10a, Wnt - 10b , Wnt- 11 , Wnt antigen comprising any suitable GPCR ligand , or any 16 , Wnt- 2 , Wnt -2b , Wnt - 3 , Wnt- 3a , Wnt - 4 , Wnt- 5a , Wnt derivative, variant or fragment thereof. Non - limiting 25 5b , Wnt- 6 , Wnt -7a , Wnt -7b , Wnt- 8a, Wnt - 8b , Wnt - 9a, Wnt examples of ligands which can be bound by a GPCR include 9b , XCL1, XCL2, Zn2 + , a -CGRP , a -ketoglutaric acid , ( - )- adrenaline , ( - ) -noradrenaline , ( lyso )phospholipid C -MSH , a -neoendorphin , B -alanine , B - CGRP , B - D -hy mediators , [des - Arg10 ] , [ des- Arg9 ]bradykinin , [des - droxybutyric acid , B - endorphin , B -MSH , B -neoendorphin , Gln14 ] ghrelin , [Hyp3 ] bradykinin , [Leulenkephalin , [Met ] B - phenylethylamine , and y -MSH . enkephalin , 12 -hydroxyheptadecatrienoic acid , 12R -HETE , 30 In some embodiments , a chimeric receptor polypeptide 12S -HETE , 12S - HPETE , 15S -HETE , 17ß - , 20 -hy comprises an integrin receptor , an integrin receptor subunit, droxy - LTB4 , 2 -arachidonoylglycerol , 2 -oleoyl - LPA , 3 -hy - or any derivative, variant or fragment thereof. Integrin droxyoctanoic acid , 5 -hydroxytryptamine , 5 -oxo - 15 -HETE , receptors are transmembrane receptors that can function as 5 -OXO -ETE , 5 -oxo -ETRE , 5 -oxo -ODE , 5S -HETE , bridges for cell - cell and cell -extracellular matrix (ECM ) 5S -HPETE , 70 , 25 - dihydroxycholesterol, acetylcholine , 35 interactions. Integrin receptors are generally formed as het ACTH , , adenosine , erodimers consisting of an a subunit and a ß subunit which 2 / intermedin , adrenomedullin , , anandamide, angio - associate non - covalently . There exist at least 18 a subunits tensin II , angiotensin III, annexin I , apelin receptor early and at least 8 B subunits . Each subunit generally comprises endogenous ligand, apelin - 13 , apelin - 17 , apelin - 36 , an extracellular region ( e . g ., ligand binding domain ) , a triggered lipoxin A4 , aspirin -triggered resolvin D1, ATP, 40 region spanning a membrane , and an intracellular region beta - defensin 4A , big dynorphin , bovine adrenal medulla ( e . g . , cytoplasmic domain ) . In some embodiments , a chime peptide 8 - 22 , bradykinin , C3a , C5a , Ca2 + , calcitonin gene ric receptor polypeptide comprises at least an extracellular related peptide, calcitonin , cathepsin G , CCK - 33 , CCK - 4 , region ( e . g ., ligand binding domain ) of an integrin subunit CCK - 8 , CCL1, CCL11 , CCL13 , CCL14 , CCL15 , CCL16 , ( e . g . , a subunit or ß subunit ) , or any derivative , variant or CCL17 , CCL19 , CCL2, CCL20 , CCL21 , CCL22 , CCL23 , 45 fragment thereof. In some embodiments , a chimeric receptor CCL24 , CCL25 , CCL26 , CCL27, CCL28 , CCL3 , CCL4 , polypeptide comprises at least a region spanning a mem CCL5 , CCL7 , CCL8 , chemerin , chenodeoxycholic acid , brane of an integrin subunit ( e . g . , a subunit or ß subunit ) , or cholic acid , corticotrophin -releasing hormone, CST- 17 , any derivative , variant or fragment thereof. In some embodi CX3CL1, CXCL1, CXCL10 , CXCL11 , CXCL12a12a ,, ments , a chimeric receptor polypeptide comprises at least an CXCL12B , CXCL13 , CXCL16 , CXCL2, CXCL3 , CXCL5, 50 intracellular region (e . g ., cytoplasmic domain ) of an integrin CXCL6 , CXCL7 , CXCL8, CXCL9, cysteinyl- leukotrienes subunit ( e . g . , a subunit or B subunit ) , or any derivative , ( CysLTs ) , nucleotides, deoxycholic acid , dihy - variant or fragment thereof. A chimeric receptor polypeptide drosphingosine - 1 - phosphate , dioleoylphosphatidic acid , comprising an integrin subunit , or any derivative , variant or dopamine , dynorphin A , dynorphin A -( 1 - 13 ) , dynorphin fragment thereof, can recruit a binding partner . In some A - ( 1 - 8 ) , dynorphin B , endomorphin - 1 , endothelin - 1 , 55 embodiments , ligand binding to a chimeric receptor com endothelin - 2 , endothelin - 3 , F2L , Free fatty acids, FSH , prising an integrin subunit, or any derivative , variant or GABA , galanin , galanin - like peptide , gastric inhibitory fragment thereof, results in a conformational change , chemi polypeptide , gastrin - 17 , gastrin - releasing peptide, ghrelin , cal modification , or combination thereof , which recruits a GHRH , glucagon , glucagon -like peptide 1 -( 7 - 36 ) amide , binding partner to the receptor. glucagon - like peptide 1 - ( 7 - 37 ), glucagon - like peptide 2 , 60 In some embodiments , a chimeric receptor polypeptide glucagon - like peptide 2 - ( 3 - 33 ) , GnRH I , GnRH II , GRP - comprises an integrin receptor a subunit , or any derivative , ( 18 - 27 ) , hCG , histamine, humanin , INSL3 , INSL5 , kallidin , variant or fragment thereof, selected from the group con kisspeptin - 10 , kisspeptin - 13 , kisspeptin - 14 , kisspeptin -54 , sisting of: al, a2 , a3 , a4 , a5 , a6 , a7 , as , a9 , alo , ali , aV , kynurenic acid , large neuromedin N , large neurotensin , aL , AM , ax , ad , aE , and allb . In some embodiments , a L - , LH , lithocholic acid , L -lactic acid , long 65 chimeric receptor polypeptide comprises an integrin recep chain carboxylic acids, LPA , LTB4, LTC4 , LTD4 , LTE4 , tor B subunit , or any derivative , variant or fragment thereof, LXA4 , Lys -[ Hyp3 ]- bradykinin , lysophosphatidylinositol, selected from the group consisting of: B1, B2 , B3 , B4 , B5 , B6 , US 9 ,856 , 497 B2 33 34 B7 , and 18 . Chimeric receptor polypeptides comprising an a herin , or any derivative , variant or fragment thereof, can subunit , a ß subunit , or any derivative , variant or fragment recruit a binding partner. In some embodiments , ligand thereof, can heterodimerize ( e . g . , a subunit dimerizing with binding to a chimeric receptor comprising a cadherin , or any a ß subunit ) to form an integrin receptor , or any derivative , derivative, variant or fragment thereof, results in a confor variant or fragment thereof . Non - limiting examples of inte - 5 mational change , chemical modification , combination grin receptors include an alß1, a2B1, a3ßi , a4B1, a5B1, thereof, which recruits a binding partner to the receptor. abßi , a7ßi, a8b1 , a9b1, a10ß1, aVB1 , alßi, ambi, Achimeric receptor polypeptide can comprise a cadherin , aXßi, adßi, allbßi , aEßi , alß2, a2ß2 , a3B2 , a4B2 , or any derivative , variant or fragment thereof, selected from a5B2 , a6B2, a7B2 , a8l2 , a982 , a1082 , aVB2 , alß2 , a classical cadherin , a desmosoma cadherin , a protocad AMB2 , aXB2, aDB2 , allbß2 , aEp2, alß3, a2B3 , a3B3 , 10 herin , and an unconventional cadherin . In some embodi a4B3 , a5B3 , abß3 , a7B3 , a8B3 , a9B3 , a1033 , aVB3, ments , a chimeric receptor polypeptide comprises a classical ALB3 , AMB3 , aXB3, aDB3 , allbB3, AEB3, a1b4, a2B4 , cadherin , or any derivative , variant or fragment thereof, a3b4, a4B4 , a5B4 , abß4 , a7B4 , a8b4 , a934 , a1084 , selected from CDH1 ( E -cadherin , epithelial) , CDH2 ( N - cad aVB4, ALB4 , AMB4 , AXB4, aDB4 , allbB4 , AEB4 , alß5 , herin , neural) , CDH12 ( cadherin 12 , type 2 , N - cadherin 2 ) , a2B5 , a3B5 , a4B5 , a5B5 , a6b5 , a7B5 , a835 , a9B5 , a10f5 , 15 and CDH3 ( P - cadherin , placental) . In some embodiments , a aVB5, ALB5 , aMß5 , aXB5 , aDB5 , allbß5, aEB5 , alp6 , chimeric receptor polypeptide comprises a desmosoma cad a2B6 , a3B6 , a436 , a536 , a6B6 , a7B6 , a8f6 , a936 , a10B6 , herin , or any derivative, variant or fragment thereof, selected aVB6 , ALB6 , AMB6 , aXB6 , aDB6 , allbß6 , AEB6 , alß7 , from desmoglein (DSG1 , DSG2 , DSG3, DSG4 ) and des a2B7 , a3B7, a4B7 , a567, a6B7 , a737 , a887 , a9B7 , a1017 , mocollin (DSC ) , DSC2, DSC3) . In some embodiments , a aVB7 , alß7, AMB7, aXB7 , aDB7 , allbß7 , aEß7, alß8 , 20 chimeric receptor polypeptide comprises a protocadherin , or a238 , 2338, a488, a5B8, a6B8 , a7B8 , a888 , A9B8 , a1088, any derivative , variant or fragment thereof , selected from aVB8 , alß8 , aMß8 , aXB8 , aDB8 , allbß8 , and aEß8 PCDH1, PCDH10 , PCDH11X , PCDH11Y, PCDH12 , receptor. A chimeric receptor polypeptide comprising an PCDH15 , PCDH17 , PCDH18 , PCDH19 , PCDH20 , PCDH7 , integrin subunit , or any derivative , variant or fragmentPCDH8, PCDH9, PCDHA1 , PCDHA10 , PCDHA11 , thereof, can dimerize with an endogenous integrin subunit 25 PCDHA12 , PCDHA13 , PCDHA2 , PCDHA3, PCDHA4 , ( e . g . , wild -type integrin subunit ) . PCDHA5, PCDHA6 , PCDHA7, PCDHAS, PCDHA9, PCD A chimeric receptor polypeptide comprising an integrin HAC1, PCDHAC2 , PCDHB1, PCDHB10 , PCDHB11 , subunit , or any derivative , variant or fragment thereof, can PCDHB12 , PCDHB13 , PCDHB14 , PCDHB15 , PCDHB16 , bind an antigen comprising any suitable integrin ligand , or PCDHB17 , PCDHB18 , PCDHB2, PCDHB3 , PCDHB4 , any derivative , variant or fragment thereof. Non - limiting 30 PCDHB5 , PCDHB6 , PCDHB7, PCDHB8 , PCDHB9, PCD examples of ligands which can be bound by an integrin HGA1, PCDHGA10 , PCDHGA11 , PCDHGA12 , PCD receptor include adenovirus penton base protein , beta - glu - HGA2 , PCDHGA3, PCDHGA4, PCDHGA5 , PCDHGA6 , can , bone sialoprotein (BSP ), Borrelia burgdorferi, Candida PCDHGA7, PCDHGAS , PCDHGA9, PCDHGB1, PCD albicans, ( CN , e. g ., CNI- IV ) , cytotactin / tenascin HGB2 , PCDHGB3, PCDHGB4, PCDHGB5 , PCDHGB6 , C , decorsin , denatured collagen , disintegrins, E - cadherin , 35 PCDHGB7 , PCDHGC3 , PCDHGC4 , PCDHGC5 , FAT, echovirus 1 receptor , epiligrin , Factor X , Fc epsilon RII FAT2, and FAT) . In some embodiments , a chimeric receptor (CD23 ) , fibrin ( Fb ), ( Fg ) , fibronectin (Fn ) , hepa - polypeptide comprises an unconventional cadherin selected rin , HIV Tat protein , iC3b , intercellular adhesion molecule from CDH4 ( R - cadherin , retinal) , CDH5 ( VE - cadherin , ( e . g . , ICAM - 1 , 2 , 3 , 4 , 5 ) , invasin , L1 cell adhesion molecule vascular endothelial) , CDH6 ( K -cadherin , ) , CDH7 (L1 - CAM ), , (LPS ) , MADCAM - 40 (cadherin 7 , type 2 ) , CDH8 ( cadherin 8 , type 2 ), CDH9 1 , - 2 (MMP ), inhibi- (cadherin 9 , type 2 , T1 - cadherin ) , CDH10 ( cadherin 10 , type tory factor (NIF ) , (OP or OPN ) , plasminogen , 2 , T2 -cadherin ), CDH11 (OB - cadherin , osteoblast) , CDH13 prothrombin , sperm fertilin , thrombospondin ( TSP ), vascu ( T- cadherin , H -cadherin , heart ), CDH15 ( M -cadherin , myo lar cell adhesion molecule 1 (VCAM - 1 ) , ( VN or tubule ), CDH16 (KSP -cadherin ) , CDH17 (LI cadherin , VTN ) , and von Willebrand factor (vWF ) . 45 - intestine ) , CDH18 ( cadherin 18 , type 2 ) , CDH19 ( cad In some embodiments , a chimeric receptor polypeptide herin 19 , type 2 ) , CDH20 ( cadherin 20 , type 2 ), CDH23 comprises a cadherin molecule, or any derivative , variant or ( cadherin 23 , neurosensory epithelium ) , CDH24 , CDH26 , fragment thereof. Cadherin molecules, which can function CDH28 , CELSR1, CELSR2 , CELSR3, CLSTN1, CLSTN2, as both ligands and receptors, refer to certain proteins CLSTN3 , DCHS1, DCHS2 , LOC389118 , PCLKC , involved in mediating cell adhesion . Cadherin molecules 50 RESDA1, and RET . generally consist of five tandem repeated extracellular chimeric receptor polypeptide comprising a cadherin , domains , a single membrane - spanning segment and a cyto - or any derivative , variant or fragment thereof , can bind an plasmic region . E - cadherin , or CDH1, for example , consists antigen comprising any suitable cadherin ligand , or any of 5 repeats in the extracellular domain , one transmembrane derivative , variant or fragment thereof. A cadherin ligand domain , and an intracellular domain . When E -cadherin is 55 can comprise, for example , another cadherin receptor ( e. g ., phosphorylated at a region of the intracellular domain , a cadherin receptor of a cell ) . adaptor proteins such as beta - catenin and p120 -catenin can In some embodiments , a chimeric receptor polypeptide bind to the receptor . In some embodiments , a chimeric comprises a catalytic receptor , or any derivative , variant or receptor polypeptide comprises at least an extracellular fragment thereof. Examples of catalytic receptors include , region of a cadherin , or any derivative , variant or fragment 60 but are not limited to , receptor tyrosine kinases (RTKs ) and thereof. In some embodiments , a chimeric receptor poly - receptor threonine /serine kinases (RTSKs ) . Catalytic recep peptide comprises at least a region spanning a membrane of tors such as RTKs and RTSKs possess certain enzymatic a cadherin , or any derivative , variant or fragment thereof. In activities. RTKs, for example , can phosphorylate substrate some embodiments, a chimeric receptor polypeptide com - proteins on tyrosine residues which can then act as binding prises at least an intracellular region ( e . g ., cytoplasmic 65 sites for adaptor proteins . RTKs generally comprise an domain ) of a cadherin , or any derivative, variant or fragment N -terminal extracellular ligand -binding domain , a single thereof. A chimeric receptor polypeptide comprising a cad transmembrane a helix , and a cytosolic C -terminal domain US 9 ,856 , 497 B2 35 36 with protein - activity . Some RTKs consist of (e .g ., MUSK receptor family such as MuSK ), or any deriva single polypeptides while some are dimers consisting of two tive, variant or fragment thereof. pairs of polypeptide chains, for example the chimeric receptor polypeptide comprising a RTK , or and some related receptors . The binding of ligands to the any derivative , variant or fragment thereof, can bind an extracellular domains of these receptors can activate the 5 antigen comprising any suitable RTK ligand , or any deriva cytosolic kinase domains, resulting in phosphorylation of tive , variant or fragment thereof. Non limiting examples of both the receptors themselves and intracellular target pro RTK ligands include growth factors, , and hor teins that propagate the signal initiated by ligand binding. In mones. Growth factors include , for example, members of the some RTKs, ligand binding induces receptor dimerization . epidermal family ( e. g ., epidermal growth Some ligands ( e. g. , growth factors such as PDGF and NGF ) 10 factor or EGF, heparin -binding EGF- like growth factor or are themselves dimers consisting of two identical polypep HB - EGF, transforming growth factor - a or TGF - a , amphi tide chains . These growth factors can directly induce regulin or AR , or EPR , , or dimerization by simultaneously binding to two different BTC , - 1 or NRG1, neuregulin - 2 or NRG2, neu receptor molecules . Other growth factors ( e . g ., such as EGF ) 15 regulin - 3 or NRG3, and neuregulin - 4 or NRG4 ), the fibro are monomers but have two distinct receptor binding sites FGF5. FGF6 , FGF7 , FGF8, FGF9 , FGF10 , FGF11, FGF12 , that can crosslink receptors . Ligand - induced dimerization FGF13 . FGF14 . FGF15 /19 . FGF16 , FGF17 , FGF18 , can result in autophosphorylation of the receptor, wherein FGF20 . FGF21 , and FGF23 ) , the vascular endothelial the dimerized polypeptide chains cross -phosphorylate one growth factor family ( e . g . , VEGF - A , VEGF- B , VEGF - C , another. Some receptors can multimerize . 20 VEGF - D , and PIGF) , and the platelet - derived growth factor In some embodiments , a chimeric receptor polypeptide family ( e .g . , PDGFA , PDGFB , PDGFC , and PDGFD ). Hor comprises at least an extracellular region (e . g ., ligand bind - mones include , for example , members of the insulin / IGF / ing domain ) of a catalytic receptor such as a RTK , or any relaxin family ( e .g ., insulin , insulin - like growth factors , derivative , variant or fragment thereof. In some embodi- relaxin family peptides including relaxinl , relaxin2 , ments , a chimeric receptor polypeptide comprises at least a 25 relaxin3 , Leydig cell - specific insulin - like peptide ( gene membrane spanning region of a catalytic receptor such as a INSL3) , early insulin - like peptide ( ELIP ) ( gene RTK , or any derivative, variant or fragment thereof. In some INSL4) , insulin - like peptide 5 ( gene INSL5 ) , and insulin embodiments , a chimeric receptor polypeptide comprises at like peptide 6 ) . least an intracellular region (e . g ., cytosolic domain ) of a In some embodiments , a chimeric receptor polypeptide catalytic receptor such as a RTK , or any derivative , variant 30 comprises at least an extracellular region ( e . g ., ligand bind or fragment thereof. A chimeric receptor polypeptide com i ng domain ) of a catalytic receptor such as an RTSK , or any prising an RTK , or any derivative, variant or fragment derivative, variant or fragment thereof. In some embodi thereof, can recruit a binding partner. In some embodiments, ments , a chimeric receptor polypeptide comprises at least a ligand binding to a chimeric receptor comprising an RTK , or membrane spanning region of a catalytic receptor such as an any derivative , variant or fragment thereof, results in a 35 RTSK , or any derivative , variant or fragment thereof. In conformational change , chemical modification , or combina - some embodiments , a chimeric receptor polypeptide com tion thereof, which recruits a binding partner to the receptor. prises at least an intracellular region ( e . g ., cytosolic domain ) In some embodiments, the chimeric receptor polypeptide of a catalytic receptor such as an RTSK , or any derivative , comprises a class I RTK ( e . g . , the variant or fragment thereof. A chimeric receptor polypeptide (EGF ) receptor family including EGFR ; the ErbB family 40 comprising an RTSK , or any derivative , variant or fragment including ErbB - 2 , ErbB - 3 , and ErbB - 4 ) , a class II RTK ( e . g . , thereof, can recruit a binding partner . In some embodiments , the insulin receptor family including INSR , IGF - 1R , and ligand binding to a chimeric receptor comprising an RTSK , IRR ) , a class III RTK ( e. g ., the platelet -derived growth or any derivative , variant or fragment thereof, results in a factor (PDGF ) receptor family including PDGFR - a , conformational change , chemical modification , or combina PDGFR - B , CSF - 1R , KIT /SCFR , and FLK2/ FLT3 ), a class 45 tion thereof, which recruits a binding partner to the receptor. IV RTK ( e . g . , the (FGF ) receptor A chimeric receptor polypeptide comprising an RTSK , or family including FGFR - 1 , FGFR - 2 , FGFR - 3 , and FGFR - 4 ), any derivative , variant or fragment thereof, can phosphory a class V RTK ( e . g . , the vascular endothelial growth factor late a substrate at serine and /or threonine residues, and may (VEGF ) receptor family including VEGFR1, VEGFR2, and select specific residues based on a consensus sequence . A VEGFR3 ) , a class VI RTK ( e . g ., the hepatocyte growth 50 chimeric receptor polypeptide can comprise a type I RTSK , factor (HGF ) receptor family including hepatocyte growth type II RTSK , or any derivative , variant or fragment thereof. factor receptor (HGFR /MET ) and RON ) , a class VII RTK . In some embodiments , a chimeric receptor polypeptide ( e . g . , the tropomyosin receptor kinase ( Trk ) receptor family comprising a type I receptor serine / threonine kinase is including TRKA , TRKB , and TRKC ), a class VIII RTK inactive unless complexed with a type II receptor. In some ( e . g ., the (Eph ) receptor family including EPHA1, 55 embodiments , a chimeric receptor polypeptide comprising a EPHA2, EPHA3 , EPHA4, EPHA5 , EPHA6 , EPHA7, type II receptor serine /threonine comprises a constitutively EPHA8 , EPHB1, EPHB2 , EPHB3, EPHB4, EPHB5, and active kinase domain that can phosphorylate and activate a EPHB6 ) , a class IX RTK (e . g. , AXL receptor family such as type I receptor when complexed with the type I receptor. A AXL , MER , and TRYO3) , a class X RTK ( e . g . , LTK type II receptor serine / threonine kinase can phosphorylate receptor family such as LTK and ALK ) , a class XI RTK 60 the kinase domain of the type I partner, causing displace ( e . g . , TIE receptor family such as TIE and TEK ) , a class XII ment of protein partners . Displacement of protein partners RTK (e . g. , ROR receptor family ROR1 and ROR2 ), a class can allow binding and phosphorylation of other proteins, for XIII RTK ( e. g ., the discoidin domain receptor ( DDR ) family example certain members of the SMAD family . A chimeric such as DDR1 and DDR2) , a class XIV RTK ( e . g ., RET receptor polypeptide can comprise a type I receptor, or any receptor family such as RET) , a class XV RTK ( e . g ., KLG 65 derivative , variant or fragment thereof , selected from the rereceptor family including PTK7) , a class XVI RTK ( e . g . , group consisting of: ALK1 (ACVRL1 ) , ALK2 ( ACVR1A ) , RYK receptor family including Ryk ), a class XVII RTK ALK3 (BMPR1A ), ALK4 ( ACVR1B ) , ALK5 ( TGFBR1 ) , US 9 ,856 , 497 B2 37 38 ALK6 (BMPR1B ), and ALK7 (ACVR1C ). A chimeric A chimeric receptor polypeptide comprising a receptor polypeptide can comprise a type II receptor, or any receptor can bind an antigen comprising any suitable derivative , variant or fragment thereof , selected from the ligand , or any derivative , variant or frag group consisting of: TGFBR2 , BMPR2, ACVR2A , ment thereof. Non - limiting examples of cytokine receptor ACVR2B , and AMHR2 (AMHR ) . In some embodiments , a 5 ligands include ( e . g . , IL -2 , IL - 3 , IL - 4 , IL - 5 , chimeric receptor polypeptide comprises a TGF -B receptor , IL -6 , IL -7 , IL - 9 , IL - 10 , IL - 11 , IL -12 , IL -13 , IL -15 , IL - 20 , or any derivative , variant or fragment thereof. IL - 21 , IL - 22 , IL -23 , IL - 27 , IL - 28 , and IL -31 ) , In some embodiments , a chimeric receptor polypeptide ( e . g . , IFN - a , IFN - B , IFN - V ) , colony stimulating factors ( e . g . , comprises a receptor which stimulates non -covalently asso , macrophage colony - stimulating factor, ciated intracellular kinases , such as a Src kinase ( e . g ., C -Src , 10 granulocyte macrophage colony - stimulating factors or GM Yes, Fyn , Fgr, Lck , Hck , Blk , Lyn , and Frk ) or a JAK kinase CSFs , and granulocyte colony - stimulating factors or ( e . g ., JAK1, JAK2, JAK3, and TYK2) rather than possess - G -CSFs ) , and ( e . g ., prolactin and ) . ing intrinsic enzymatic activity , or any derivative , variant or In some embodiments , a chimeric receptor polypeptide fragment thereof. These include the cytokine receptor super - comprises a death receptor, a receptor containing a death family such as receptors for cytokines and polypeptide 15 domain , or any derivative, variant or fragment thereof. hormones . Cytokine receptors generally contain an N -ter - Death receptors are often involved in regulating apoptosis minal extracellular ligand -binding domain , transmembrane and . Death receptors include members of the a helices, and a C - terminal cytosolic domain . The cytosolic TNF receptor family such as TNFR1, , DR4 domains of cytokine receptors are generally devoid of any (also known as TRAIL receptor 1 or TRAILR1) and DR5 known catalytic activity . Cytokine receptors instead can 20 (also known as TRAIL receptor 2 or TRAILR2 ) . In some function in association with non - receptor kinases (e . g ., tyro - embodiments , a chimeric receptor polypeptide comprises at sine kinases or threonine /serine kinases ), which can be least an extracellular region ( e . g . , ligand binding domain ) of activated as a result of ligand binding to the receptor. In a death receptor, or any derivative, variant or fragment some embodiments , a chimeric receptor polypeptide com - thereof . In some embodiments , a chimeric receptor poly prises at least an extracellular region ( e . g . , ligand binding 25 peptide comprises at least a membrane spanning region of a domain ) of a catalytic receptor that non - covalently associ- death receptor, or any derivative , variant or fragment ates with an intracellular kinase ( e . g . , a cytokine receptor ), thereof . In some embodiments , a chimeric receptor poly or any derivative , variant or fragment thereof. In some peptide comprises at least an intracellular region ( e . g . , embodiments , a chimeric receptor polypeptide comprises at cytosolic ) domain of a death receptor, or any derivative , least a membrane spanning region of a catalytic receptor that 30 variant or fragment thereof . A chimeric receptor polypeptide non - covalently associates with an intracellular kinase ( e . g ., comprising a death receptor , or any derivative, variant or a cytokine receptor ), or any derivative, variant or fragment fragment thereof, can undergo receptor oligomerization in thereof. In some embodiments , a chimeric receptor poly - response to ligand binding , which in turn can result in the peptide comprises at least an intracellular region ( e. g ., recruitment of specialized adaptor proteins and activation of cytosolic domain ) of a catalytic receptor that non - covalently 35 signaling cascades , such as caspase cascades . In some associates with an intracellular kinase ( e . g . , a cytokine embodiments , a chimeric receptor polypeptide comprises a receptor) , or any derivative , variant or fragment thereof. A death receptor, or any derivative , variant or fragment chimeric receptor polypeptide comprising a catalytic recep - thereof, results in a conformational change , chemical modi tor that non - covalently associates with an intracellular fication , or combination thereof, which recruits a binding kinase , or any derivative , variant or fragment thereof, can 40 partner to the receptor. recruit a binding partner. In some embodiments , ligand chimeric receptor polypeptide comprising a death binding to a chimeric receptor comprising a catalytic recep - receptor can bind an antigen comprising any suitable ligand tor that non - covalently associates with an intracellular of a death receptor, or any derivative , variant or fragment kinase, or any derivative, variant or fragment thereof, results thereof. Non - limiting examples of ligands bound by death in a conformational change , chemicalmodification , or com - 45 receptors include TNFa , , and TNF - related apop bination thereof, which recruits a binding partner to the tosis - inducing ligand ( TRAIL ) . receptor In some embodiments , a chimeric receptor polypeptide In some embodiments , a chimeric receptor polypeptide comprises an , or any derivative , variant or comprises a cytokine receptor, for example a type I cytokine fragment thereof. Immune receptors include members of the receptor or a type II cytokine receptor, or any derivative , 50 immunoglobulin superfamily (IgSF ) which share structural variant or fragment thereof . In some embodiments , the features with immunoglobulins, e . g . , a domain known as an chimeric receptor polypeptide comprises an immunoglobulin domain or fold . IgSF members include , but receptor ( e . g . , IL - 2R , IL - 3R , IL - 4R , IL -5R , IL -6R , IL - 7R , are not limited to , cell surface antigen receptors , co - recep IL - 9R , IL - 11R , IL - 12R , IL - 13R , IL - 15R , IL - 21R , IL - 23R , tors and costimulatory molecules of the immune system , and IL -27R , and IL -31R ), a colony stimulating factor receptor 55 molecules involved in to lymphocytes. ( e . g ., , CSF - 1R , CSF - 2R , GM -CSFR , In some embodiments, a chimeric receptor polypeptide and G - CSFR ) , a hormone receptor/ comprises at least an extracellular region ( e . g ., ligand bind ( e . g . , , , and lep - ing domain ) of an immune receptor , or any derivative , tin receptor ) , or any derivative, variant or fragment thereof. variant or fragment thereof. In some embodiments , a chi In some embodiments , the chimeric receptor polypeptide 60 meric receptor polypeptide comprises at least a region comprises a type II cytokine receptor, or any derivative , spanning a membrane of an immune receptor , or any deriva variant or fragment thereof. In some embodiments , the tive , variant or fragment thereof. In some embodiments , a chimeric receptor polypeptide comprises an chimeric receptor polypeptide comprises at least an intrac receptor (e . g ., IFNAR1 , IFNAR2, and IFNGR ), an interleu - ellular region (e .g . , cytoplasmic domain ) of an immune kin receptor ( e . g . , IL - 10R , IL - 20R , IL - 22R , and IL - 28R ) , a 65 receptor, or any derivative , variant or fragment thereof. A receptor (also called platelet tissue factor ) , or chimeric receptor polypeptide comprising an immune recep any derivative, variant or fragment thereof. tor, or any derivative, variant or fragment thereof , can recruit US 9 , 856 ,497 B2 39 40 a binding partner. In some embodiments , ligand binding to edit the sequence of a nucleic acid ( e . g ., a gene and /or gene a chimeric receptor comprising an immune receptor, or any product) . In some embodiments , the actuator moiety com derivative , variant or fragment thereof, results in a confor - prises a DNA nuclease such as an engineered ( e . g . , pro mational change , chemical modification , or combination grammable or targetable) DNA nuclease to induce genome thereof, which recruits a binding partner to the receptor. 5 editing of a target DNA sequence . In some embodiments , the In some embodiments , a chimeric receptor polypeptide actuator moiety comprises a RNA nuclease such as an comprises a cell surface antigen receptor such as a engineered ( e . g . , programmable or targetable ) RNA nucle receptor ( TCR ) , a B cell receptor (BCR ) , or any derivative , ase to induce editing of a target RNA sequence . In some variant or fragment thereof. T cell receptors generally com - embodiments , the actuator moiety has reduced or minimal prise two chains , either the TCR - alpha and - beta chains or 10 nuclease activity. An actuator moiety having reduced or the TCR -delta and - gamma chains. A chimeric receptor minimal nuclease activity can regulate expression and /or polypeptide comprising a TCR , or any derivative , variant or activity of a gene by physical obstruction of a target poly fragment thereof , can bind a major histocompatibility com - nucleotide or recruitment of additional factors effective to plex (MHC ) protein . B cell receptors generally comprises a suppress or enhance expression of the target polynucleotide . membrane bound immunoglobulin and a 15 In some embodiments , the actuator moiety comprises a moiety . A chimeric receptor comprising a BCR , or any nuclease - null DNA binding protein derived from a DNA derivative , variant or fragment thereof, can bind a cognate nuclease that can induce transcriptional activation or repres BCR antigen . In some embodiments , a chimeric receptor sion of a target DNA sequence . In some embodiments , the polypeptide comprises at least an immunoreceptor tyrosine actuator moiety comprises a nuclease -null RNA binding based activation motif (ITAM ) found in the cytoplasmic 20 protein derived from a RNA nuclease that can induce domain of certain immune receptors. In some embodiments , transcriptional activation or repression of a target RNA a chimeric receptor polypeptide comprises at least an immu - sequence . In some embodiments , the actuator moiety is a noreceptor tyrosine -based inhibition motif (ITIM ) found in nucleic acid - guided actuator moiety . In some embodiments , the cytoplasmic domain of certain immune receptors . Chi - the actuator moiety is a DNA - guided actuator moiety . In meric receptor polypeptides comprising ITAM and / or ITIM 25 some embodiments , the actuator moiety is an RNA - guided domains can be phosphorylated following ligand binding to actuator moiety . An actuator moiety can regulate expression an antigen interacting domain . The phosphorylated regions or activity of a gene and / or edit a nucleic acid sequence , can serve as docking sites for other proteins involved in whether exogenous or endogenous . immune . Any suitable nuclease can be used in an actuator moiety . The antigen interacting domain of a chimeric receptor 30 Suitable nucleases include , but are not limited to , CRISPR polypeptide can bind a membrane bound antigen , for associated (Cas ) proteins or Cas nucleases including type I example an antigen bound to the extracellular surface of a CRISPR -associated (Cas ) polypeptides , type II CRISPR cell ( e . g ., a target cell ) . In some embodiments , the antigen associated (Cas ) polypeptides, type III CRISPR -associated interacting domain binds a non -membrane bound antigen , ( Cas ) polypeptides , type IV CRISPR - associated (Cas ) poly for example an extracellular antigen that is secreted by a cell 35 peptides, type V CRISPR - associated (Cas ) polypeptides, ( e. g ., a target cell) or an antigen located in the cytoplasm of and type VI CRISPR -associated ( Cas ) polypeptides; a cell . Antigens ( e . g . , membrane bound and non -membrane finger nucleases (ZFN ) ; transcription activator - like effector bound ) can be associated with a disease such as a viral, nucleases ( TALEN ) ; meganucleases; RNA -binding proteins bacterial , and / or parasitic infection ; inflammatory and /or (RBP ) ; CRISPR - associated RNA binding proteins; recom autoimmune disease ; or neoplasm such as a cancer and / or 40 binases , flippases ; transposases , Argonaute ( Ago ) proteins tumor. Cancer antigens, for example , are proteins produced (e . g ., prokaryotic Argonaute (pAgo ), archaeal Argonaute by tumor cells that can elicit an immune response , particu - (aAgo ), and eukaryotic Argonaute ( eAgo ) ) ; any derivative larly a T - cell mediated immune response . The selection of thereof, any variant thereof; and any fragment thereof. the antigen binding portions of a chimeric receptor poly - The regulation of genes can be of any gene of interest . It peptide can depend on the particular type of cancer antigen 45 is contemplated that genetic homologues of a gene described to be targeted . In some embodiments , the tumor antigen herein are covered . For example , a gene can exhibit a certain comprises one or more antigenic cancer epitopes associated identity and /or to genes disclosed herein . There with a malignant tumor. Malignant tumors can express a fore , it is contemplated that a gene that exhibits or exhibits number of proteins that can serve as target antigens for an about 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 81 % , 82 % , immune attack . The antigen interaction domains can bind to 50 83 % , 84 % , 85 % , 86 % , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , cell surface signals , extracellular matrix ( ECM ) , paracrine 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , 99 % , or 100 % homology signals , juxtacrine signals , endocrine signals , autocrine sig (at the nucleic acid or protein level ) can be modified . It is nals, signals that can trigger or control genetic programs in also contemplated that a gene that exhibits or exhibits about cells , or any combination thereof. In some embodiments , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 81 % , 82 % , 83 % , interactions between the cell signals that bind to the recom - 55 84 % , 85 % , 86 % , 87 % 88 % , 89 % , 90 % , 91 % , 92 % , 93 % . binant chimeric receptor polypeptides involve a cell -cell 94 % , 95 % , 96 % , 97 % , 98 % , 99 % , or 100 % identity ( at the interaction , cell -soluble chemical interaction , and cell -ma - nucleic acid or protein level ) can be modified . trix or microenvironment interaction . In some embodiments , the actuator moiety comprises a A gene modulating polypeptide (GMP ) of a chimeric CRISPR - associated ( Cas ) protein or a Cas nuclease which receptor polypeptide can comprise an actuator moiety linked 60 functions in a non - naturally occurring CRISPR ( Clustered to a cleavage recognition site . The actuator moiety can Regularly Interspaced Short Palindromic Repeats ) /Cas comprise a nuclease ( e . g . , DNA nuclease and / or RNA nucle - ( CRISPR -associated ) system . In bacteria , this system can ase ) , modified nuclease ( e . g . , DNA nuclease and / or RNA provide adaptive immunity against foreign DNA (Barran nuclease ) that is nuclease - deficient or has reduced nuclease gou , R . , et al, “ CRISPR provides acquired resistance against activity compared to a wild -type nuclease, a derivative 65 viruses in prokaryotes ," Science ( 2007 ) 315 : 1709 - 1712 ; thereof, a variant thereof, or a fragment thereof. The actuator Makarova , K . S . , et al, “ Evolution and classification of the moiety can regulate expression or activity of a gene and / or CRISPR -Cas systems, ” Nat Rev Microbiol (2011 ) 9 :467 US 9 ,856 ,497 B2 41 42 477 ; Garneau , J. E . , et al, “ The CRISPR / Cas bacterial or to edit a nucleic acid sequence. In some embodiments , the immune system cleaves bacteriophage and plasmid DNA ,” actuator moiety comprises a nuclease -null DNA binding Nature (2010 ) 468 :67 -71 ; Sapranauskas , R . , et al, “ The protein derived from a DNA nuclease that can induce Streptococcus thermophilus CRISPR / Cas system provides transcriptional activation or repression of a target DNA immunity in Escherichia coli , ” Nucleic Acids Res ( 2011) 5 sequence . In some embodiments , the actuator moiety com 39 : 9275 - 9282 ). prises a nuclease -null RNA binding protein derived from a In a wide variety of organisms including diverse mam RNA nuclease that can induce transcriptional activation or mals , animals , plants , and yeast, a CRISPR / Cas system ( e . g . , repression of a target RNA sequence. For example , an modified and / or unmodified ) can be utilized as a genome actuator moiety can comprise a Cas protein which lacks engineering tool. A CRISPR / Cas system can comprise a 10 cleavage activity . guide nucleic acid such as a guide RNA ( GRNA ) complexed Any suitable CRISPR /Cas system can be used . A with a Cas protein for targeted regulation of gene expression CRISPR / Cas system can be referred to using a variety of and / or activity or nucleic acid editing . An RNA - guided Cas naming systems. Exemplary naming systems are provided in protein ( e . g . , a Cas nuclease such as a Cas9 nuclease ) can Makarova , K . S . et al, “ An updated evolutionary classifica specifically bind a target polynucleotide ( e . g ., DNA ) in a 15 tion of CRISPR - Cas systems, ” Nat Rev Microbiol ( 2015 ) sequence - dependent manner. The Cas protein , if possessing 13 : 722 - 736 and Shmakov, S . et al, “ Discovery and Func nuclease activity , can cleave the DNA (Gasiunas , G . , et al, tional Characterization of Diverse Class 2 CRISPR -Cas “ Cas9 -crRNA ribonucleoprotein complex mediates specific Systems, ” Mol Cell ( 2015 ) 60 : 1 - 13 . A CRISPR /Cas system DNA cleavage for adaptive immunity in bacteria ,” Proc Natl can be a type I, a type II, a type III , a type IV , a type V , a Acad SciUSA (2012 ) 109 : E2579 -E2 86 ; Jinek , M ., et al , “ A 20 type VI system , or any other suitable CRISPR / Cas system . programmable dual- RNA - guided DNA endonuclease in ACRISPR / Cas system as used herein can be a Class 1 , Class adaptive bacterial immunity, " Science ( 2012 ) 337 :816 - 821 ; 2 , or any other suitably classified CRISPR / Cas system . Class Sternberg, S . H ., et al, “ DNA interrogation by the CRISPR 1 or Class 2 determination can be based upon the genes RNA - guided endonuclease Cas9, " Nature (2014 ) 507 :62 ; encoding the effector module. Class 1 systems generally Deltcheva , E ., et al, “ CRISPR RNA maturation by trans - 25 have a multi - subunit rRNA - effector complex , whereas encoded small RNA and host factor RNase III ,” Nature Class 2 systems generally have a single protein , such as ( 2011) 471 :602 -607 ), and has been widely used for pro - Cas9 , Cpfi , C2c1 , C2c2 , C2c3 or a crRNA - effector com grammable genome editing in a variety of organisms and plex . A Class 1 CRISPR / Cas system can use a complex of model systems ( Cong, L ., et al, “ Multiplex genome engi - multiple Cas proteins to effect regulation . A Class 1 neering using CRISPR Cas systems, " Science (2013 ) 339 : 30 CRISPR /Cas system can comprise , for example , type I ( e . g . , 819 -823 ; Jiang , W ., et al, “ RNA - guided editing of bacterial I , IA , IB , IC , ID , IE , IF , IU ) , type III ( e . g ., III , IIIA , IIIB , using CRISPR -Cas systems, ” Nat. Biotechnol. IIIC , IIID ) , and type IV ( e . g . , IV , IVA , IVB ) CRISPR / Cas ( 2013 ) 31 : 233 - 239 ; Sander , J. D . & Joung, J. K , “ CRISPR - type. A Class 2 CRISPR /Cas system can use a single large Cas systems for editing, regulating and targeting genomes , ” Cas protein to effect regulation . A Class 2 CRISPR / Cas Nature Biotechnol. ( 2014 ) 32 : 347 -355 ) . 35 systems can comprise , for example , type II ( e . g . , II , IIA , IIB ) In some cases, the Cas protein is mutated and /or modified and type V CRISPR /Cas type. CRISPR systems can be to yield a nuclease deficient protein or a protein with complementary to each other, and /or can lend functional decreased nuclease activity relative to a wild - type Cas units in trans to facilitate CRISPR locus targeting . FIG . 15 protein . A nuclease deficient protein can retain the ability to shows an illustration adapted from FIG . 2 of Makarova , K . bind DNA , but may lack or have reduced nucleic acid 40 S . et al , “ An updated evolutionary classification of CRISPR cleavage activity . An actuator moiety comprising a Cas Cas systems, ” Nat Rev Microbiol ( 2015 ) 13 :722 -736 pro nuclease ( e .g ., retaining wild -type nuclease activity , having viding architectures of the genomic loci for subtypes of reduced nuclease activity , and /or lacking nuclease acitivity ) CRISPR -Cas systems. can function in a CRISPR /Cas system to regulate the level An actuator moiety comprising a Cas protein can be a and / or activity of a target gene or protein ( e. g . , decrease , 45 Class 1 or a Class 2 Cas protein . A Cas protein can be a type increase , or elimination ) . The Cas protein can bind to a I, type II , type III , type IV , type V Cas protein , or type VI Cas target polynucleotide and prevent transcription by physical protein . A Cas protein can comprise one or more domains . obstruction or edit a nucleic acid sequence to yield non Non - limiting examples of domains include , guide nucleic functional gene products . acid recognition and / or binding domain , nuclease domains In some embodiments , the actuator moiety comprises a 50 ( e . g . , DNase or RNase domains , RuvC , HNH ) , DNA binding Cas protein that forms a complex with a guide nucleic acid , domain , RNA binding domain , helicase domains , protein such as a guide RNA . In some embodiments , the actuator protein interaction domains , and dimerization domains . A moiety comprises a Cas protein that forms a complex with guide nucleic acid recognition and / or binding domain can a single guide nucleic acid , such as a single guide RNA interact with a guide nucleic acid . A nuclease domain can ( sgRNA ) . In some embodiments , the actuator moiety com - 55 comprise catalytic activity for nucleic acid cleavage . A prises a RNA - binding protein (RBP ) optionally complexed nuclease domain can lack catalytic activity to prevent with a guide nucleic acid , such as a guide RNA ( e . g ., nucleic acid cleavage. A Cas protein can be a chimeric Cas SgRNA ), which is able to form a complex with a Cas protein . protein that is fused to other proteins or polypeptides. A Cas FIG . 3A illustrates schematically a system comprising a protein can be a chimera of various Cas proteins, for chimeric receptor polypeptide in which the actuator moiety 60 example , comprising domains from different Cas proteins . comprises an RNA - binding protein 300a optionally com - Non - limiting examples of Cas proteins include c2c1 , plexed with a guide nucleic acid ( e . g ., sgRNA ) . Upon C2c2 , c2c3 , Cas? , Cas1B , Cas2 , Cas3 , Cast , Cas5 , Cas5e release from the RNA - binding protein (RBP ), for example (CasD ), Cash , Casbe , Cas6f, Cas7, Cas8a , Cas8al, Cas8a2 , by dissociation of the guide nucleic acid from the RBP or Cas8b , Cas8c, Cas9 (Csnl or Csx12) , Cas10 , Cas10d , cleavage of the cleavage recognition site 300c , the guide 65 Cas10 , Cas1Od , CasF, CasG , CasH , Cpfl, Csyl, Csy2 , nucleic acid can form a complex with a Cas protein 300b Csy3 , Csel (CasA ) , Cse2 ( CasB ) , Cse3 ( CasE ) , Cse4 which is operable to regulate gene expression and / or activity (CasC ) , Csci , Csc2, Csa5 , Csn2 , Csm2, Csm3, Csm4 , US 9 ,856 ,497 B2 43 44 Csm5, Csm , Cmrl, Cmr3 , Cmr4 , Cmr5, Cmr6 , Csbl, proteobacterium , Legionella pneumophila , Parasutterella Csb2 , Csb3 , Csx17 , Csx14 , Csx10 , Csx16 , CsaX , Csx3 , excrementihominis , Wolinella succinogenes , and Francisella Csx1, Csx15 , Csfl , Csf2 , Csf3 , Csf4 , and Cul966 , and novicida . homologs or modified versions thereof. A Cas protein as used herein can be a wildtype or a A Cas protein can be from any suitable organism . Non - 5 modified form of a Cas protein . A Cas protein can be an limiting examples include Streptococcus pyogenes , Strepto - active variant, inactive variant, or fragmentof a wild type or coccus thermophilus, Streptococcus sp ., Staphylococcus modified Cas protein . A Cas protein can comprise an amino aureus, Nocardiopsis dassonvillei , Streptomyces pristinae acid change such as a deletion , insertion , substitution , vari spiralis , Streptomyces viridochromo genes , Streptomyces a nt, mutation , fusion , chimera , or any combination thereof viridochromogenes , Streptosporangium roseum , Streptospo - relative to a wild -type version of the Cas protein . A Cas rangium roseum , AlicyclobacHlus acidocaldarius, Bacillus protein can be a polypeptide with at least about 5 % , 10 % , pseudomycoides, Bacillus selenitireducens, Exiguobacte 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 91 % , 92 % , rium sibiricum , Lactobacillus delbrueckii, Lactobacillus 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , 99 % , or 100 % sequence salivarius, Microscilla marina , Burkholderiales bacterium , 16 identity or sequence similarity to a wild type exemplary Cas Polaromonas naphthalenivorans, Polaromonas sp . , Cro - protein . A Cas protein can be a polypeptide with at most cosphaera watsonii , Cyanothece sp ., Microcystis aerugi- about 5 % , 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , nosa , Pseudomonas aeruginosa , Synechococcus sp ., Aceto 90 % , 100 % sequence identity and /or sequence similarity to halobium arabaticum , Ammonifex degensii, a wild type exemplary Cas protein . Variants or fragments Caldicelulosiruptor becscii, Candidatus Desulforudis , 20 can comprise at least about 5 % , 10 % , 20 % , 30 % , 40 % , 50 % , Clostridium botulinum , Clostridium difficile , Finegoldia 60 % , 70 % , 80 % , 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , magna , Natranaerobius thermophilus, Pelotomaculum ther- 97 % , 98 % , 99 % , or 100 % sequence identity or sequence mopropionicum , Acidithiobacillus caldus , Acidithiobacillus similarity to a wild type or modified Cas protein or a portion ferrooxidans , Allochromatium vinosum , Marinobacter sp ., thereof. Variants or fragments can be targeted to a nucleic Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudo - 25 acid locus in complex with a guide nucleic acid while alteromonas haloplanktis, Ktedonobacter racemifer, Metha - lacking nucleic acid cleavage activity. nohalobium evestigatum , Anabaena variabilis , Nodularia A Cas protein can comprise one or more nuclease spumigena, Nostoc sp ., Arthrospira maxima , Arthrospira domains, such as DNase domains . For example, a Cas9 platensis , Arthrospira sp . , Lyngbya sp ., Microcoleus chtho protein can comprise a RuvC - like nuclease domain and /or iphe 30 an HNH - like nuclease domain . The RuvC and HNH noplastes, Oscillatoria sp ., Petrotoga mobilis , Thermosipho 30 domains can each cut a different strand of double - stranded africanus, Acaryochloris marina , Leptotrichia shahii, and DNA to make a double -stranded break in the DNA . A Cas Francisella novicida . In some aspects , the organism is protein can comprise only one nuclease domain ( e . g . , Cpfi Streptococcus pyogenes ( S . pyogenes ). In some aspects , the comprises RuvC domain but lacks HNH domain ) . organism is Staphylococcus aureus ( S . aureus ) . In some 35, A Cas protein can comprise an amino acid sequence aspects , the organism is Streptococcus thermophilus ( S . having at least about 5 % , 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , thermophilus ). 70 % , 80 % , 90 % , 91 % 92 % 93 % , 94 % , 95 % , 96 % , 97 % , A Cas protein can be derived from a variety of bacterial 98 % . 99 % , or 100 % sequence identity or sequence similarity including , but not limited to , Veillonella atypical, to a nuclease domain ( e . g . , RuvC domain , HNH domain ) of Fusobacterium nucleatum , Filifactor alocis , Solobacterium 40 a wild -type Cas protein . moorei , Coprococcus catus, Treponema denticola , Pep A Cas protein can be modified to optimize regulation of toniphilus duerdenii , Catenibacterium mitsuokai, Strepto - gene expression . A Cas protein can be modified to increase coccus mutans , Listeria innocua , Staphylococcus pseudin - or decrease nucleic acid binding affinity , nucleic acid bind termedius, Acidaminococcus intestine , Olsenella uli, ing specificity , and / or enzymatic activity . Cas proteins can Oenococcus kltaharae , Bifidobacterium bifidum , Lactoba - 45 also be modified to change any other activity or property of cillus rhamnosus, Lactobacillus gasseri, Finegoldia magna , the protein , such as stability . For example , one or more Mycoplasma mobile , Mycoplasma gallisepticum , Myco nuclease domains of the Cas protein can be modified , plasma ovipneumoniae , Mycoplasma canis, Mycoplasma deleted , or inactivated , or a Cas protein can be truncated to synoviae , Eubacterium rectale , Streptococcus thermophilus, remove domains that are not essential for the function of the Eubacterium dolichum , Lactobacillus coryniformis subsp . 50 protein or to optimize ( e . g ., enhance or reduce ) the activity Torquens , Ilyobacter polytropus, Ruminococcus albus, of the Cas protein for regulating gene expression . Akkermansia muciniphila , Acidothermus cellulolyticus , Bifi - A Cas protein can be a fusion protein . For example, a Cas dobacterium longum , Bifidobacterium dentium , Corynebac protein can be fused to a cleavage domain , an epigenetic terium diphtheria , Elusimicrobium minutum , Nitratifractor modification domain , a transcriptional activation domain , or salsuginis, Sphaerochaeta globus, Fibrobacter succino - 55 a transcriptional repressor domain . A Cas protein can also be genes subsp . Succinogenes , Bacteroides fragilis , Capnocy fused to a heterologous polypeptide providing increased or tophaga ochracea , Rhodopseudomonas palustris , Prevotella decreased stability . The fused domain or heterologous poly micans , Prevotella ruminicola , Flavobacterium columnare, peptide can be located at the N - terminus, the C - terminus, or Aminomonas paucivorans , Rhodospirillum rubrum , Candi- internally within the Cas protein . datus Puniceispirillum marinum , Verminephrobacter eise - 60 Cas protein can be provided in any form . For example , niae, Ralstonia syzygii, Dinoroseobacter shibae , Azospiril- a Cas protein can be provided in the form of a protein , such lum , Nitrobacter hamburgensis , Bradyrhizobium , Wolinella as a Cas protein alone or complexed with a guide nucleic succinogenes, Campylobacter jejuni subsp . Jejuni, Helico acid . A Cas protein can be provided in the form of a nucleic bacter mustelae, Bacillus cereus, Acidovorax ebreus, acid encoding the Cas protein , such as an RNA ( e . g . , Clostridium perfringens, Parvibaculum lavamentivorans, 65 messenger RNA (mRNA ) ) or DNA . The nucleic acid encod Roseburia intestinalis , Neisseria meningitidis , Pasteurella ing the Cas protein can be codon optimized for efficient multocida subsp . Multocida, Sutterella wadsworthensis , translation into protein in a particular cell or organism . US 9 ,856 , 497 B2 45 46 Nucleic acids encoding Cas proteins can be stably inte - CRISPR RNA (crRNA ) recognition sequence within a grated in the genome of the cell . Nucleic acids encoding Cas double - stranded DNA but not a double - strand break . Such a proteins can be operably linked to a promoter active in the nickase can cleave the complementary strand or the non cell. Nucleic acids encoding Cas proteins can be operably complementary strand , but may not cleave both . If all of the linked to a promoter in an expression construct. Expression 5 nuclease domains of a Cas protein ( e . g . , both RuvC and constructs can include any nucleic acid constructs capable of HNH nuclease domains in a Cas9 protein ; RuvC nuclease directing expression of a gene or other nucleic acid sequence domain in a Cpfl protein ) are deleted or mutated , the of interest ( e . g . , a Cas gene ) and which can transfer such a resulting Cas protein can have a reduced or no ability to nucleic acid sequence of interest to a target cell . cleave both strands of a double - stranded DNA . An example In some embodiments , a Cas protein is a dead Cas protein . 10 of a mutation that can convert a Cas9 protein into a nickase A dead Cas protein can be a protein that lacks nucleic acid is a D10A (aspartate to alanine at position 10 of Cas9 ) cleavage activity . mutation in the RuvC domain of Cas9 from S . pyogenes. A Cas protein can comprise a modified form of a wild type H939A ( histidine to alanine at amino acid position 839 ) or Cas protein . The modified form of the wild type Cas protein H840A (histidine to alanine at amino acid position 840 ) in can comprise an amino acid change ( e . g . , deletion , insertion , 15 the HNH domain of Cas9 from S. pyogenes can convert the or substitution ) that reduces the nucleic acid - cleaving activ - Cas9 into a nickase . An example of a mutation that can ity of the Cas protein . For example , the modified form of the convert a Cas9 protein into a dead Cas9 is a D10A (aspartate Cas protein can have less than 90 % , less than 80 % , less than to alanine at position 10 of Cas9 ) mutation in the Ruvc 70 % , less than 60 % , less than 50 % , less than 40 % , less than domain and H939A (histidine to alanine at amino acid 30 % , less than 20 % , less than 10 % , less than 5 % , or less than 20 position 839 ) or H840A (histidine to alanine at amino acid 1 % of the nucleic acid -cleaving activity of the wild - type Cas position 840 ) in the HNH domain of Cas9 from S . pyogenes . protein ( e . g ., Cas9 from S . pyogenes ) . The modified form of A dead Cas protein can comprise one or more mutations Cas protein can have no substantial nucleic acid - cleaving relative to a wild - type version of the protein . The mutation activity . When a Cas protein is a modified form that has no can result in less than 90 % , less than 80 % , less than 70 % , substantial nucleic acid - cleaving activity , it can be referred 25 less than 60 % , less than 50 % , less than 40 % , less than 30 % , to as enzymatically inactive and / or “ dead ” (abbreviated by less than 20 % , less than 10 % , less than 5 % , or less than 1 % “ d ” ) . A dead Cas protein ( e . g . , d?as , dCas9 ) can bind to a of the nucleic acid - cleaving activity in one or more of the target polynucleotide but may not cleave the target poly - plurality of nucleic acid - cleaving domains of the wild -type nucleotide . In some aspects , a dead Cas protein is a dead Cas protein . The mutation can result in one or more of the Cas9 protein . 30 plurality of nucleic acid - cleaving domains retaining the A dCas9 polypeptide can associate with a single guide ability to cleave the complementary strand of the target RNA (SORNA ) to activate or repress transcription of target nucleic acid but reducing its ability to cleave the non DNA . SORNAS can be introduced into cells expressing the complementary strand of the target nucleic acid . The muta engineered chimeric receptor polypeptide . In some cases , tion can result in one or more of the plurality of nucleic such cells contain one or more different sgRNAs that target 35 acid - cleaving domains retaining the ability to cleave the the same nucleic acid . In other cases , the sgRNAs target non - complementary strand of the target nucleic acid but different nucleic acids in the cell . The nucleic acids targeted reducing its ability to cleave the complementary strand of by the guide RNA can be any that are expressed in a cell the target nucleic acid . The mutation can result in one or such as an immune cell . The nucleic acids targeted may be more of the plurality of nucleic acid - cleaving domains a gene involved in immune cell regulation . In some embodi- 40 lacking the ability to cleave the complementary strand and ments , the nucleic acid is associated with cancer. The nucleic the non - complementary strand of the target nucleic acid . The acid associated with cancer can be a cell cycle gene , cell residues to be mutated in a nuclease domain can correspond response gene, apoptosis gene , or phagocytosis gene . The to one or more catalytic residues of the nuclease . For recombinant guide RNA can be recognized by a CRISPR example , residues in the wild type exemplary S . pyogenes protein , a nuclease -null CRISPR protein , variants thereof, or 45 Cas9 polypeptide such as Asp10 , His840 , Asn854 and derivatives thereof. Asn856 can be mutated to inactivate one or more of the Enzymatically inactive can refer to a polypeptide that can plurality of nucleic acid - cleaving domains ( e . g ., nuclease bind to a nucleic acid sequence in a polynucleotide in a domains ) . The residues to be mutated in a nuclease domain sequence - specific manner, but may not cleave a target poly - of a Cas protein can correspond to residues Asp10 , His840 , nucleotide. An enzymatically inactive site -directed polypep - 50 Asn854 and Asn856 in the wild type S . pyogenes Cas9 tide can comprise an enzymatically inactive domain (e . g . polypeptide, for example , as determined by sequence and /or nuclease domain ) . Enzymatically inactive can refer to no s tructural alignment. activity . Enzymatically inactive can refer to substantially no As non - limiting examples , residues D10 , G12 , G17 , activity . Enzymatically inactive can refer to essentially no E762 , H840 , N854 , N863, H982 , H983, A984, D986 , and / or activity . Enzymatically inactive can refer to an activity less 55 A987 ( or the corresponding mutations of any of the Cas than 1 % , less than 2 % , less than 3 % , less than 4 % , less than proteins ) can be mutated . For example , e . g . , D10A , G12A , 5 % , less than 6 % , less than 7 % , less than 8 % , less than 9 % , G17A , E762A , H840A , N854A , N863A , H982A , H983A , or less than 10 % activity compared to a wild - type exemplary A984A , and/ or D986A . Mutations other than alanine sub activity ( e . g ., nucleic acid cleaving activity , wild -type Cas9 s titutions can be suitable . A D10A mutation can be combined activity ). 60 with one or more ofH840A , N854A , or N856A mutations to One or a plurality of the nuclease domains (e . g ., Ruvc , produce a Cas9 protein substantially lacking DNA cleavage HNH ) of a Cas protein can be deleted or mutated so that they activity ( e . g ., a dead Cas9 protein ) . A H840A mutation can are no longer functional or comprise reduced nuclease be combined with one or more of D10A , N854A , or N856A activity . For example , in a Cas protein comprising at least mutations to produce a site - directed polypeptide substan two nuclease domains ( e . g . , Cas9 ) , if one of the nuclease 65 tially lacking DNA cleavage activity . A N854A mutation can domains is deleted or mutated , the resulting Cas protein , be combined with one or more of H840A , D10A , or N856A known as a nickase , can generate a single- strand break at a mutations to produce a site - directed polypeptide substan US 9 ,856 , 497 B2 47 48 tially lacking DNA cleavage activity . A N856A mutation can ing in HDR . Descriptions of nickases are found , be combined with one or more of H840A , N854A , or D10A e . g ., in Ramirez et al . , Nucl Acids Res, 2012 , 40 ( 12 ) :5560 - 8 ; mutations to produce a site - directed polypeptide substan - Kim et al. , Genome Res, 2012 , 22 ( 7 ) : 1327 - 33 . In some tially lacking DNA cleavage activity . embodiments , a ZFN binds a polynucleotide ( e .g ., DNA In some embodiments , a Cas protein is a Class 2 Cas 5 and /or RNA ) but is unable to cleave the polynucleotide . protein . In some embodiments , a Cas protein is a type II Cas In some embodiments , the cleavage domain of an actuator protein . In some embodiments , the Cas protein is a Cas9 moiety comprising a ZFN comprises a modified form of a protein , a modified version of a Cas9 protein , or derived wild type cleavage domain . The modified form of the from a Cas9 protein . For example , a Cas9 protein lacking cleavage domain can comprise an amino acid change ( e . g . , cleavage activity . In some embodiments , the Cas9 protein is 10 deletion , insertion , or substitution ) that reduces the nucleic a Cas9 protein from S. pyogenes ( e . g . , SwissProt accession acid - cleaving activity of the cleavage domain . For example , number ( 99ZW2) . In some embodiments , the Cas9 protein the modified form of the cleavage domain can have less than is a Cas9 from S . aureus ( e . g ., SwissProt accession number 90 % , less than 80 % , less than 70 % , less than 60 % , less than JYRUA5 ) . In some embodiments , the Cas9 protein is a 50 % , less than 40 % , less than 30 % , less than 20 % , less than modified version of a Cas9 protein from S . pyogenes or S . 15 10 % , less than 5 % , or less than 1 % of the nucleic acid Aureus. In some embodiments , the Cas9 protein is derived cleaving activity of the wild - type cleavage domain . The from a Cas9 protein from S . pyogenes or S . Aureus. For modified form of the cleavage domain can have no substan example , a S . pyogenes or S . Aureus Cas9 protein lacking t ial nucleic acid - cleaving activity . In some embodiments , the cleavage activity . cleavage domain is enzymatically inactive . Cas9 can generally refer to a polypeptide with at least 20 In some embodiments , an actuator moiety comprises a about 5 % , 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , “ TALEN ” or “ TAL -effector nuclease. ” TALENs refer to 90 % , 100 % sequence identity and / or sequence similarity to engineered transcription activator - like effector nucleases a wild type exemplary Cas9 polypeptide ( e .g ., Cas9 from S . that generally contain a central domain of DNA -binding pyogenes ) . Cas9 can refer to a polypeptide with at most tandem repeats and a cleavage domain . TALENs can be about 5 % , 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 25 produced by fusing a TAL effector DNA binding domain to 90 % , 100 % sequence identity and / or sequence similarity to a DNA cleavage domain . In some cases , a DNA -binding a wild type exemplary Cas9 polypeptide ( e . g ., from S . tandem repeat comprises 33 - 35 amino acids in length and pyogenes ) . Cas9 can refer to the wildtype or a modified form contains two hypervariable amino acid residues at positions of the Cas9 protein that can comprise an amino acid change 12 and 13 that can recognize at least one specific DNA base such as a deletion , insertion , substitution , variant, mutation , 30 pair . A transcription activator- like effector ( TALE ) protein fusion , chimera, or any combination thereof. can be fused to a nuclease such as a wild - type or mutated In some embodiments , an actuator moiety comprises a Foki endonuclease or the catalytic domain of Fokl. Several “ zinc finger nuclease ” or “ ZFN . ” ZFNs refer to a fusion mutations to FokI have been made for its use in TALENs, between a cleavage domain , such as a cleavage domain of which , for example , improve cleavage specificity or activity . Fokl, and at least one zinc finger motif ( e . g . , at least 2 , 3 , 4 , 35 Such TALENs can be engineered to bind any desired DNA or 5 zinc finger motifs ) which can bind polynucleotides such sequence . TALENs can be used to generate gene modifica as DNA and RNA . The heterodimerization at certain posi tions ( e . g ., nucleic acid sequence editing ) by creating a tions in a polynucleotide of two individual ZFNs in certain double - strand break in a target DNA sequence , which in orientation and spacing can lead to cleavage of the poly - turn , undergoes NHEJ or HDR . In some cases, a single nucleotide . For example , a ZFN binding to DNA can induce 40 stranded donor DNA repair template is provided to promote a double - strand break in the DNA . In order to allow two HDR . Detailed descriptions of TALENs and their uses for cleavage domains to dimerize and cleave DNA , two indi gene editing are found , e . g . , in U . S . Pat . Nos. 8 , 440, 431 ; vidual ZFNs can bind opposite strands of DNA with their 8 ,440 ,432 ; 8 ,450 ,471 ; 8 ,586 ,363 ; and 8 ,697 ,853 ; Scharen C - termini at a certain distance apart. In some cases , linker b et al. , Curr Gene Ther , 2013 , 13 ( 4 ) :291 - 303 ; Gaj et al. , sequences between the zinc finger domain and the cleavage 45 NatMethods , 2012 , 9( 8 ): 805 - 7 ; Beurdeley et al. , Nat Com domain can require the 5 ' edge of each to be mun , 2013 , 4 : 1762 ; and Joung and Sander , Nat Rev MolCell separated by about 5 - 7 base pairs . In some cases , a cleavage Biol, 2013 , 14 ( 1 ) : 49 - 55 . domain is fused to the C - terminus of each zinc finger I n some embodiments , a TALEN is engineered for domain . Exemplary ZFNs include , but are not limited to , reduced nuclease activity . In some embodiments , the nucle those described in Urnov et al. , Nature Reviews Genetics , 50 ase domain of a TALEN comprises a modified form of a wild 2010 , 11 :636 -646 ; Gaj et al. , Nat Methods, 2012 , 9 ( 8 ) : 805 type nuclease domain . The modified form of the nuclease 7 ; U . S . Pat. Nos . 6 ,534 , 261; 6 ,607 , 882 ; 6 ,746 ,838 ; 6 ,794 , domain can comprise an amino acid change ( e . g . , deletion , 136 ; 6 ,824 , 978 ; 6 , 866 , 997 ; 6 , 933 , 113 ; 6 , 979 , 539 ; 7 , 013 , insertion , or substitution ) that reduces the nucleic acid 219 ; 7 ,030 , 215 ; 7 ,220 ,719 ; 7 ,241 ,573 ; 7 ,241 ,574 ; 7 ,585 , cleaving activity of the nuclease domain . For example , the 849; 7 ,595 ,376 ; 6 , 903 , 185 ; 6 ,479 ,626 ; and U . S . Application 55 modified form of the nuclease domain can have less than Publication Nos . 2003 /0232410 and 2009 /0203140 . 90 % , less than 80 % , less than 70 % , less than 60 % , less than In some embodiments, an actuator moiety comprising a 50 % , less than 40 % , less than 30 % , less than 20 % , less than ZFN can generate a double - strand break in a target poly - 10 % , less than 5 % , or less than 1 % of the nucleic acid nucleotide , such as DNA . A double - strand break in DNA can cleaving activity of the wild - type nuclease domain . The result in DNA break repair which allows for the introduction 60 modified form of the nuclease domain can have no substan of gene modification ( s ) ( e . g ., nucleic acid editing ) . DNA tial nucleic acid -cleaving activity . In some embodiments , the break repair can occur via non - homologous end joining nuclease domain is enzymatically inactive . ( NHEJ) or homology -directed repair (HDR ). In HDR , a In some embodiments , the transcription activator- like donor DNA repair template that contains homology arms effector ( TALE ) protein is fused to a domain that can flanking sites of the target DNA can be provided . In some 65 modulate transcription and does not comprise a nuclease . In embodiments , a ZFN is a zinc finger nickase which induces some embodiments , the transcription activator - like effector site -specific single - strand DNA breaks or nicks, thus result - (TALE ) protein is designed to function as a transcriptional US 9 ,856 , 497 B2 49 50 activator. In some embodiments , the transcription activator Epstein - Barr virus R transactivator ( Rta ) and are described like effector ( TALE ) protein is designed to function as a in Chavez et al. , Nat Methods, 2015 , 12 (4 ): 326 - 328 and U .S . transcriptional repressor. For example , the DNA -binding Patent App . Publ. No . 20140068797 . Non - limiting exem domain of the transcription activator - like effector (TALE ) plary repression domains include the KRAB (Krüppel protein can be fused ( e . g . , linked ) to one or more transcrip - 5 associated box ) domain of Kox1 , the Mad mSIN3 interac tional activation domains, or to one or more transcriptional tion domain ( SID ) , ERF repressor domain (ERD ) , and are repression domains. Non - limiting examples of a transcrip described in Chavez et al ., Nat Methods , 2015 , 12 ( 4 ) : 326 tional activation domain include a herpes simplex VP16 328 and U . S . Patent App . Publ. No . 20140068797 . An activation domain and a tetrameric repeat of the VP16 actuator moiety can also be fused to a heterologous poly activation domain , e . g . , a VP64 activation domain . A non - 10 peptide providing increased or decreased stability . The fused limiting example of a transcriptional repression domain domain or heterologous polypeptide can be located at the includes a Krtippel- associated box domain . N - terminus , the C - terminus , or internally within the actuator In some embodiments , an actuator moiety comprises a moiety . meganuclease . Meganucleases generally refer to rare - cut An actuator moiety can comprise a heterologous poly ting endonucleases or homing endonucleases that can be 15 peptide for ease of tracking or purification , such as a highly specific . Meganucleases can recognize DNA target fluorescent protein , a purification tag , or an epitope tag . sites ranging from at least 12 base pairs in length , e . g ., from Examples of fluorescent proteins include green fluorescent 12 to 40 base pairs , 12 to 50 base pairs , or 12 to 60 base pairs proteins ( e . g ., GFP , GFP - 2 , tagGFP , turboGFP, eGFP , Emer in length . Meganucleases can be modular DNA - binding ald , Azami Green , Monomeric Azami Green , CopGFP, nucleases such as any fusion protein comprising at least one 20 AceGFP , ZsGreen1 ), yellow fluorescent proteins ( e .g ., YFP, catalytic domain of an endonuclease and at least one DNA eYFP, Citrine , Venus, Y Pet , PhiYFP , ZsYellowl) , blue fluo binding domain or protein specifying a nucleic acid target rescent proteins ( e . g . eBFP , eBFP2 , Azurite , mKalamal, sequence . The DNA -binding domain can contain at least one GFPuv, Sapphire, T - sapphire) , cyan fluorescent proteins motif that recognizes single - or double - stranded DNA . The ( e . g . eCFP , Cerulean , CyPet, AmCyanl , Midoriishi - Cyan ) , meganuclease can be monomeric or dimeric . In some 25 red fluorescent proteins (mKate , mKate2 , mPlum , DsRed embodiments , the meganuclease is naturally - occurring monomer, mCherry , mRFP1 , DsRed - Express, DsRed2 , ( found in nature ) or wild -type , and in other instances , the DsRed -Monomer , HcRed - Tandem , HcRedi , AsRed2 , meganuclease is non - natural, artificial, engineered , syn eqFP611 , mRaspberry , mStrawberry, Jred ) , orange fluores thetic, rationally designed , or man -made . In some embodi- cent proteins (mOrange , mKO , Kusabira -Orange , Mono ments , the meganuclease of the present disclosure includes 30 meric Kusabira -Orange , mTangerine , tdTomato ) , and any an I - Crel meganuclease , 1 -Ceul meganuclease , I -Msol other suitable fluorescent protein . Examples of tags include meganuclease , I - Scel meganuclease , variants thereof, glutathione - S - transferase (GST ) , chitin binding protein derivatives thereof, and fragments thereof. Detailed descrip - (CBP ) , maltose binding protein , thioredoxin ( TRX ) , poly tions of useful meganucleases and their application in gene (NANP ) , tandem affinity purification ( TAP ) tag , , AcV5 , editing are found , e . g. , in Silva et al. , Curr Gene Ther, 2011, 35 AU1, AU5, E , ECS , E2 , FLAG , hemagglutinin (HA ), nus, 11 ( 1 ) :11 - 27 ; Zaslavoskiy et al . , BMC Bioinformatics , 2014 , Softag 1 , Softag 3 , Strep , SBP, Glu -Glu , HSV , KT3, S , SI, 15 :191 ; Takeuchi et al ., Proc Natl Acad Sci USA , 2014 , 17 , V5 , VSV - G , histidine (His ) , biotin carboxyl carrier 111 (11 ) : 4061 -4066 , and U . S . Pat . Nos. 7 ,842 ,489 ; 7 ,897 , protein (BCCP ), and . 372 ; 8 ,021 , 867; 8 , 163 ,514 ; 8 ,133 ,697 ; 8 ,021 , 867 ; 8 , 119 , The cleavage recognition site of a GMP can be flanked by 361 ; 8 , 119 ,381 ; 8 , 124 , 36 ; and 8 , 129, 134 . 40 the antigen interacting domain and the actuator moiety in In some embodiments, the nuclease domain of a mega - some configurations of a chimeric receptor polypeptide . The nuclease comprises a modified form of a wild type nuclease actuator moiety can be released from the GMP by cleavage domain . The modified form of the nuclease domain can of the recognition site by a cleavage moiety . A cleavage comprise an amino acid change ( e . g ., deletion , insertion , or moiety can recognize and /or cleave a cleavage recognition substitution ) that reduces the nucleic acid - cleaving activity 45 site , for example, when in proximity to the cleavage recog of the nuclease domain . For example , the modified form of nition site . A cleavage moiety can comprise a polypeptide the nuclease domain can have less than 90 % , less than 80 % , sequence . The cleavage moiety can form a portion of the less than 70 % , less than 60 % , less than 50 % , less than 40 % , chimeric adaptor polypeptide . The cleavage moiety can less than 30 % , less than 20 % , less than 10 % , less than 5 % , form the N -terminus , C - terminus , or an internal portion of or less than 1 % of the nucleic acid - cleaving activity of the 50 the chimeric adaptor polypeptide. In some embodiments , the wild -type nuclease domain . Themodified form of the nucle cleavage moiety is complexed to the chimeric adaptor ase domain can have no substantial nucleic acid - cleaving polypeptide. The cleavage moiety can be complexed to the activity . In some embodiments , the nuclease domain is N -terminus , C - terminus , or an internal portion of the chi enzymatically inactive . In some embodiments , a meganucle meric adaptor polypeptide . FIG . 3B shows an exemplary ase can bind DNA but cannot cleave the DNA . 55 arrangement of the various components of a subject system . In some embodiments , the actuator moiety is fused to one The cleavage recognition site 302b of a GMP is flanked by or more transcription repressor domains , activator domains, the antigen interacting domain 301 and the actuator moiety epigenetic domains , recombinase domains , transposase 302a , and the cleavage moiety 304 forms a portion of a domains, flippase domains, nickase domains, or any com - chimeric adaptor polypeptide 303 . bination thereof. The activator domain can include one or 60 FIGS . 4A - D illustrate schematically the release of an more tandem activation domains located at the carboxyl actuator moiety from a GMP . FIG . 4A shows the binding of terminus of the enzyme. In other cases, the actuator moiety an antigen to a transmembrane chimeric receptor polypep includes one or more tandem repressor domains located at tide . The transmembrane chimeric receptor polypeptide the carboxyl terminus of the protein . Non - limiting exem - comprises an extracellular region having an antigen inter plary activation domains include GAL4 , herpes simplex 65 acting domain 401 and an intracellular region comprising a activation domain VP16 , VP64 ( a tetramer of the herpes GMP. The GMP includes an actuator moiety 402a linked to simplex activation domain VP16 ) , NF -KB p65 subunit, a cleavage recognition site 402b . In response to antigen US 9 ,856 , 497 B2 51 binding, the receptor is modified by phosphorylation 403 in cus ); superfamily PD proteases , e . g . , family C46 including the intracellular region of the receptor ( FIG . 4B ). Following Hedgehog protein (Drosophila melanogaster ); superfamily receptor modification ( e . g ., phosphorylation ), an adaptor PE proteases , e . g ., family P1 including DmpA aminopepti protein comprising a receptor binding moiety is recruited to dase (Ochrobactrum anthropi) ; others proteases, e . g ., family the receptor as shown in FIG . 4C . The receptor comprises a 5 C7, C8 , C21, C23 , C27 , C36 , C42 , C53 and C75 . Additional cleavage moiety 404 ; the cleavage moiety may be com - proteases include serine proteases, e . g ., those of superfamily plexed with the adaptor or linked , for example by a peptide SB , e . g ., families S8 and 953 including subtilisin (Bacillus bond and /or peptide linker , to the receptor binding moiety . licheniformis ); those of superfamily SC , e. g ., families S9 , When in proximity to the cleavage recognition site , the S10 , S15 , S28 , S33 , and S37 including prolyl oligopeptidase cleavage moiety can cleave the recognition site to release the 10 (Sus scrofa ) ; those of superfamily SE , e . g . , families S11 , actuator moiety from the GMP as shown in FIG . 4D . Upon S12 , and S13 including D - Ala - D - Ala peptidase C (Escheri release , the actuatormoiety can enter the nucleus to regulate chia coli ) ; those of superfamily SF , e . g ., families S24 and the expression and / or activity of a target gene or edit a S26 including signal peptidase I ( Escherichia coli ) ; those of nucleic acid sequence . FIGS. 4E - H show an analogous Superfamily SJ, e . g . , families S16 , S50 , and S69 including system wherein receptor modification comprises a confor - 15 Ion - A peptidase ( Escherichia coli ) ; those of Superfamily mational change . In some embodiments , the adaptor protein SK , e . g . , families S14 , S41, and S49 including Clp protease is tethered to the membrane (e . g ., as a membrane bound (Escherichia coli ); those of Superfamily SO , e . g . , families protein ) . S74 including Phage K1F endosialidase CIMCD self - cleav In some embodiments , the cleavage moiety only cleaves ing protein ( Enterobacteria phage K1F ) ; those of superfam the recognition site when in proximity to the cleavage 20 ily SP , e . g. , family S59 including nucleoporin 145 (Homo recognition site . The cleavage recognition site can comprise sapiens ); those of superfamily SR , e . g . , family $60 includ a polypeptide sequence that is a recognition sequence of a ing Lactoferrin (Homo sapiens) ; those of superfamily SS , protease . The cleavage moiety can comprise protease activ families S66 including murein tetrapeptidase LD - carboxy ity which recognizes the polypeptide sequence . A cleavage peptidase ( Pseudomonas aeruginosa ) ; those of superfamily moiety comprising protease activity can be a protease , or 25 ST, e . g . , families S54 including rhomboid - 1 (Drosophila any derivative, variant or fragment thereof. A protease refers melanogaster ); those of superfamily PA , e . g . , families Si, to any enzyme that performs proteolysis , in which polypep - S3 , S6 , S7, S29 , S30 , S31 , S32, S39, S46 , S55 , S64 , S65 , tides are cleaved into smaller polypeptides or amino acids. and S75 including Chymotrypsin A (Bos taurus) ; those of Various proteases are suitable for use as a cleavage moiety . superfamily PB , e . g ., families S45 and 563 including peni Some proteases can be highly promiscuous such that a wide 30 cillin G acylase precursor ( Escherichia coli ) ; those of super range of protein substrates are hydrolysed . Some proteases family PC , e . g . , families S51 including dipeptidase E ( Es can be highly specific and only cleave substrates with a cherichia coli ); those of superfamily PE , e. g ., families P1 certain sequence , e . g ., a cleavage recognition sequence or including DmpA aminopeptidase (Ochrobactrum anthropi) ; peptide cleavage domain . In some embodiments , the cleav those unassigned , e . g ., families S48 , S62, S68 , 371 , 372 , age recognitions site comprises multiple cleavage recogni - 35 S79, and S81 threonine proteases , e . g . , those of superfamily tion sequences , and each cleavage recognition sequence can PB clan , e . g . , families T1 , T2, T3 , and T6 including archaean be recognized by the same or different cleavage moiety , ß component ( Thermoplasma acidophilum ) ; comprising protease activity ( e . g ., protease ) . Sequence -spe - and those of superfamily PE clan , e . g . , family T5 including cific proteases that can be used as cleavage moieties include, ornithine acetyltransferase (Saccharomyces cerevisiae) ; but are not limited to , superfamily CA proteases , e . g ., 40 aspartic proteases , e . g . , BACE1, BACE2 ; cathepsin D ; families C1, C2, C6 , C10 , C12 , C16 , C19 , C28 , C31 , C32 , cathepsin E ; chymosin ; napsin - A ; nepenthesin ; pepsin ; plas C33 , C39 , C47 , C51, C54 , C58 , C64, C65 , C66 , C67 , C70 , mepsin ; presenilin ; ; and HIV - 1 protease, and metallo C71 , C76 , 278 , C83 , C85 , C86 , C87 , C93 , C96 , C98 , and proteinases , e . g ., exopeptidases , metalloexopeptidases; C101, including papain (Carica papaya ), bromelain endopeptidases , and metalloendopeptidases. A cleavage rec (Ananas comosus ), cathepsin K (liverwort ) and calpain 45 ognition sequence ( e . g ., polypeptide sequence ) can be rec (Homo sapiens) ; superfamily CD proteases, e . g . , family ognized by any of the proteases disclosed herein . C11 , C13 , C14 , C25, C50 , C80 , and C84 : such as caspase - 1 In some embodiments , the cleavage recognition site com ( Rattus norvegicus ) and separase ( Saccharomyces cerevi - prises a cleavage recognition sequence ( e . g ., polypeptide siae ) ; superfamily CE protease , e . g . , family C5 , C48 , C55 , sequence or peptide cleavage domain ) that is recognized by C57 , C63 , and C79 including adenain (human adenovirus 50 a protease selected from the group consisting of: achro type 2 ) ; superfamily CF proteases, e . g ., family C15 includ - mopeptidase , aminopeptidase , ancrod , angiotensin convert ing pyroglutamyl- peptidase I (Bacillus amyloliquefaciens) ; ing enzyme, bromelain , calpain , calpain I, calpain II , car superfamily CL proteases , e . g . , family C60 and C82 includ boxypeptidase A , carboxypeptidase B , carboxypeptidase G , ing sortase A (Staphylococcus aureus ) ; superfamily CM carboxypeptidase P , carboxypeptidase W , carboxypeptidase proteases, e .g . family C18 including hepatitis C virus pep - 55 Y , caspase 1, caspase 2 , caspase 3, caspase 4 , caspase 5 , tidase 2 ( hepatitis C virus ); superfamily CN proteases, e . g . , , caspase 7 , caspase 8 , caspase 9 , caspase 10 , family C9 including sindbis virus - type nsP2 peptidase ( sind - caspase 11 , caspase 12 , caspase 13 , cathepsin B , cathepsin bis virus ) ; superfamily CO proteases, e . g ., family C40 C , cathepsin D , cathepsin E , cathepsin G , cathepsin H , including dipeptidyl- peptidase VI (Lysinibacillus sphaeri- cathepsin L , chymopapain , chymase , chymotrypsin , clostri cus ) ; superfamily CP proteases , e . g . , family C97 including 60 pain , collagenase , complement C1r , complement Cls , DeSI- 1 peptidase (Mus musculus ) ; superfamily PA pro - complement Factor D , complement factor I , cucumisin , teases, e . g ., family C3 , C4 , C24 , C30 , C37 , C62 , C74 , and dipeptidyl peptidase IV , elastase (leukocyte ) , elastase ( pan C99 including TEV protease ( Tobacco etch virus) ; super creatic ), endoproteinase Arg - C , endoproteinase Asp - N , family PB proteases , e . g . , family C44 , C45 , C59, C69, C89 , endoproteinase Glu - C , endoproteinase Lys- C , enterokinase , and C95 including amidophosphoribosyltransferase precur - 65 factor Xa , ficin , furin , granzyme A , granzyme B , HIV sor (homo sapiens ) ; superfamily PC proteases , families C26 , Protease , IGase , kallikrein tissue , leucine aminopeptidase and C56 including Y - glutamyl hydrolase (Rattus norvegi ( general ) , leucine aminopeptidase ( cytosol) , leucine amino US 9 , 856 ,497 B2 53 54 peptidase (microsomal ) ,matrix metalloprotease , teinase from Aspergillus oryzae , proteinase 3 , proteinase A , aminopeptidase , neutrase , papain , pepsin , plasmin , proli - proteinase K , protein C , pyroglutamate aminopeptidase , dase , pronase E , prostate specific antigen , protease alkalo - rennin , rennin , streptokinase , subtilisin , thermolysin , throm philic from Streptomyces griseus , protease from Aspergillus, bin , tissue plasminogen activator, trypsin , tryptase and uro protease from Aspergillus saitoi, protease from Aspergillus 5 kinase . sojae , protease ( B . licheniformis ) ( alkaline or alcalase ), Table 1 lists exemplary proteases and associated recog protease from Bacillus polymyxa , protease from Bacillus sp , nition sequences that can be used in systems of the disclo protease from Rhizopus sp . , protease S , , pro sure . TABLE 1 Exemplary proteases and associated recognition sequences Protease name Synonyms Recognition sequence Arg - C Arginyl peptidase, Endoproteinase Arg - C , Tissue R - x kallikrein Asp - N Endoproteinase Asp - N , Peptidyl- Asp X - D metalloendopeptidase Asp - N (N Endoproteinase Asp - N , Peptidyl - Asp x - [DE ] terminal Glu ) metalloendopeptidase BNPS or 3 - Bromo- 3 -methyl - 2 - ( 2 - nitrophenylthio ) -3H - indole , W - x NCS/ urea BNPS - skatol, N -chlorosuccinimide / urea Caspase - 1 ICE , Interleukin -1ß - Converting Enzyme [FLWY ] - x - [AHT ]- D {DEKPQR } ( SEQ ID NO : 61 ) Caspase - 10 Flice2 , Mch4 I - E - A - D - x ( SEQ ID NO : 62 ) Caspase - 2 Ich - 1 , Nedd2 D - V - A - D - {DEKPQR } ( SEQ ID NO : 63 ) or D - E - H D - { DEKPQR } (SEQ ID NO : 64 ) Caspase - 3 Apopain , CPP32 , Yama D - M - Q - D - {DEKPQR } ( SEQ ID NO : 65 ) or D - E - V - D { DEKPQR } (SEQ ID NO : 66 ) Caspase - 4 ICE ( rel )II , Ich - 2 , TX L - E - V - D - DEKPQR } ( SEQ ID NO : 67 ) or (LW ) - E - H - D { DEKPQR } (SEQ ID NO : 68 ) Caspase- 5 ICE ( rel) III, TY [LW ] - E - H - D - x (SEQ ID NO : 69 ) Caspase - 6 Mch2 V - E -[ HI ] - D - {DEKPQR } (SEQ ID NO : 70 ) Caspase- 7 CMH -1 , ICE -LAP3 , Mch -3 D - E - V - D - {DEKPQR } (SEQ ID NO : 71 ) Caspase- 8 FLICE , MASH , Mch5 [ IL ] - E - T - D - {DEKPQR } (SEQ ID NO : 72 ) Caspase - 9 ICE - Lap6 , Mch6 L - E - H - D - x (SEQ ID NO : 73 ) Chymotrypsin [FY ]- { P } or W - {MP } Chymotrypsin [FLY ] - { P } or W - {MP } or ( low M - { PY } or H - { DMPW } specificity ) Clostripain Clostridiopeptidase B R - x CNBr Cyanogen bromide M - x CNBr (methyl - Cyanogen bromide M - x or x - C Cys ) CNBr (with Cyanogen bromide [MW ]- X acids ) Enterokinase Enteropeptidase [DE ]( 4 ) -K -x (SEQ ID NO : 74 ) Factor Xa Coagulation factor Xa [ AFGILTVM ] - [DE ] - G - R - X (SEQ ID NO : 75 ) Formic acid D - x Glu - C ( AmAc Endoproteinase Glu - C , V8 protease, Glutamyl E - x buffer ) endopeptidase Glu - C (Phos Endoproteinase Glu - C , V8 protease , Glutamyl [DE ]- X buffer ) endopeptidase Granzyme B Cytotoxic T -lymphocyte proteinase 2 , Granzyme- 2 , 1- E - P - D - x (SEQ ID NO : 76 ) GranzymeB , Lymphocyte protease , SECT, T -cell serine protease 1 - 3E HRV3C Human rhinovirus 3C protease , Picornain 3C , Protease L - E - V - L - F - Q - G - P ( SEQ ID protease 3C NO : 77 ) Hydroxylamine Hydroxylammonium N - G Iodosobenzoic 2 -Iodosobenzoic acid W - x acid Lys - C Endoproteinase Lys- C , Lysyl endopeptidase K - x Lys - N Endoproteinase Lys- N , Peptidyl- Lys x - K metalloendopeptidase, Armillaria mellea neutral proteinase US 9 , 856 , 497 B2 55 56 TABLE 1 -continued Exemplary proteases and associated recognition sequences Protease name Synonyms Recognition sequence Lys - N (Cys Endoproteinase Lys - N , Peptidyl - Lys x - [CK ] modified ) metalloendopeptidase , Armillaria mellea neutral proteinase Mild acid D - P hydrolysis NBS ( long N - Bromosuccinimide [ HWY ] - x exposure ) NBS ( short N -Bromosuccinimide [WY ]- X exposure ) NTCB 2 -Nitro - 5 -thiocyanatobenzoic acid , 2 -Nitro - 5 X - C thiocyanobenzoic acid Pancreatic Pancreatopeptidase E , Elastase - 1 [ AGSV ] -X elastase Pepsin A Pepsin {HKR }- { P } - { R } - [ FLWY] { P } (SEQ ID NO : 78 ) or { HKR }- { P }- [FLWY ] - x - { P } ( SEQ ID NO : 79 ) Pepsin A ( low Pepsin { HKR } - {P } - {R } - [FL ] -{ P } specificity ) ( SEQ ID NO : 80 ) or HKR } - {P } - [FL ] - x - { P } (SEQ ID NO : 81 ) Prolyl Prolyl oligopeptidase , Post -proline cleaving enzyme [HKR ] - P - { P } endopeptidase Proteinase K Endopeptidase K , Peptidase K [ AEFILTVWY] - X TEV protease Tobacco etch virus protease , Nuclear- inclusion -a E - x - x - Y - x - Q - [GS ] (SEQ ID endopeptidase NO : 82) Thermolysin Thermophilic - bacterial protease { DE } - [ AFILMV ] - { P } Thrombin Factor Ila x - x - G - R - G - X (SEQ ID NO : 83) or ( AFGILTVW ] [ AFGILTVW ] - P - R - { DE } {DE } (SEQ ID NO : 84 ) Trypsin Trypsin - 1 X - [KR ]- { P } or W - K - P or M R - P But not : CD ] - K - D or C - K - [HY ] or C - R - K or R - R - [HR ] Trypsin ( Arg K - {P } blocked ) Trypsin ( Cys [RKC ]- { P } modified ) Trypsin ( Lys R - { P } blocked )

Proteases selected for use as cleavage moieties can be selected based on desired characteristics such as peptide bond selectivity , activity at certain pHs, molecular mass , etc . The properties of exemplary proteases are provided in Table TABLE 2 Exemplary proteases and protease characteristics Peptide bond pH Molecular Accession Protease EC no . Class selectivity optimum mass ( kDa ) no . Endoproteinase Trypsin (bovine ) 3. 4 .21 . 4 serine P1- P11 - (P1 = Lys, 8 .0 -9 . 0 23. 5 P007605 Arg ) Chymotrypsin 3 . 4 ..21 . 1 serine P ,- P !- (21sP1 = 7 . 5 - 8 . 5 25 P007665P007665 (bovine ) aromatic, P , ' = nonspecific ) Endoproteinase 3. 4 . 24 . 33 metallo P ,- Asp - ( and - P ,- 6. 0 -8 . 0 ñ Asp - N cysteic acid ) (Pseudomonas fragi) Endoproteinase serine - Arg - P1 8 .0 - 8 . 5 n . a . Arg - C (mouse submaxillary gland ) US 9 , 856 , 497 B2 57 58 TABLE 2 -continued Exemplary proteases and protease characteristics Peptide bond pH Molecular Accession |4Protease EC no . Class selectivity optimum mass (kDa ) no . Endoproteinase 3 . 4 .21 . 19 serine -Glu - P , 1 - (and 8 . 0 27 P041885 Glu - C ( V8 - Asp -P11 -) (2 ) protease ) ( Staphylococcus aureus ) Endoproteinase 3 . 4 .21 . 50 serine - Lys - P , 1 8 . 0 30NR 33R S77957P Lys - C ( Lysobacter enzymogenes) Pepsin (porcine ) 3 . 4 . 23. 1 aspartic P . - P . - (P1 = 2 . 0 - 4 . 0 34 . 5 P00791S hydrophobic preferred ) Thermolysin 3 . 4 . 24 . 27 metallo P Leu, 75 .. 0 - 9 . 00 37 . 5 P008005 ( Bacillus Phe , Ile , Val, Met, 7. 0 - 9 . 0 37. 5 POO8OOS thermoproteolyticus ) Ala ) Elastase (porcine ) 3. 4 .21 . 36 serine P ,- P , '- (P1 = 7. 8 -8 .5 25 . 9 P007725 uncharged , 7.8 - 8 . 5 25, 9 P007728 nonaromatic ) Papain ( Carica 3. 4. 22 . 2 cysteine P1- P1 ? - (P1 = Arg, 6 . 0 - 7 . 0 23 P007845 papaya ) Lys preferred ) Proteinase K 3 . 4 . 21. 64 serine P . - P , 1 - (P1 = 7 . 5 - 12 . 0 18 . 5 P06873. 5 ( Tritirachium aromatic , album ) hydrophobic preferred ) Subtilisin 3. 4. 21 . 62 serine P . -P , 1 - (P1 = 7 . 0 - 11 . 0 309 27. 32 P041895 (Bacillus subtilis ) neutral/ acidic preferred ) Clostripain 3. 4 .22 .8 cysteine - Arg - P ,- (P1 = Pro 7 .1 -7 . 6 59 P09870S ( endoproteinase preferred ) Arg - C ) ( Clostridium histolyticum ) !11111111111111 Exopeptidase Carboxypeptidase 3. 4 . 17 .1 metallo P , -P , 1- (P , cannot 7. 0 -8 . 0 34 . 5 P00730 $ A (bovine ) be Arg, Lys , Pro ) Carboxypeptidase 3 .4 . 17. 2 metallo P1- P1 ? - (P1 = Lys , 7 .0 - 9 . 0 34 . 6 P007325 B (porcine ) Arg ) 7. 0 - 9 . 0 34. 6 P007328 Carboxypeptidase serine P . - P , 4 . 0 - 5 . 0 n . a . P (Penicillium ( nonspecific ) janthinellum ) Carboxypeptidase 3 .4 . 16 . 5 serine P1-P1 . 5 . 5 - 6 . 5 61 P007295 Y (yeast ) (nonspecific ) Cathepsin C 3 . 4 . 14 . 1 cysteine X - P , - P / ? 55 . 5 210 n .. a . (removes amino terminal dipeptide ) Acylamino -acid - 3. 4 . 19 . 1 serine AC- P1- P1 - (P1 = 7 . 5 804 360P P192059 + releasing enzyme Ser , Ala , Met (porcine ) preferred ) Pyroglutamate 3 . 4 . 19 . 3 cysteine P , - P , 1 - (P , = 7 . 0 - 9 . 0 70 - 80B n . a . aminopeptidase 5 - oxoproline or (bovine ) pyroglutamate )

50 In some embodiments , the cleavage recognition site com - In an illustrative example shown in FIG . 13A , a chimeric prises a first portion of an intein sequence that reacts with the receptor polypeptide comprises an actuator moiety 1301 second portion of the intein sequence to release the actuator covalently linked ( e .g ., at its N - terminus or C - terminus ) via moiety . A heterologous split intein system can be used to a peptide bond to a first portion of the intein sequence 1302 , facilitate release of the actuator moiety from the chimeric receptor polypeptide . The actuator moiety can be covalentlyC 55 which comprises an N -terminal intein . The actuator moiety linked to the first portion of the intein sequence . The actuator N -terminal intein fusion can be contacted with a second moiety can be linked via its N - terminus or C - terminus to the portion of the intein sequence 1303 comprising a C -terminal first portion of the intein sequence . The second portion of the intein as shown in FIG . 13B , for example a second portion intein sequence can be a part of the chimeric adaptor of the intein sequence linked to an adaptor polypeptide . This polypeptide. The second portion of the intein sequence can 60 contacting of the first and second portion of the intein serve as a cleavage moiety . The first portion or second sequences can result in a site specific cleavage ( e . g . , at a site portion of the intein sequence can be the N -terminal intein , between thethe actuator moiety and thethe NN --terminal intein ) as the C -terminal intein , or any other suitable portion of an shown in FIG . 13C , thereby releasing the actuator moiety as intein that can facilitate release of the actuator moiety . The shown in FIG . 13D . In an alternative configuration shown in intein sequences can be from any suitable source. The first 65 FIGS . 13E - H , the actuator moiety is linked and /or com and second portion can be from the same or different sources plexed to the adaptor polypeptide rather than the receptor ( e . g . , organism , protein ) . polypeptide. In another illustrative example , an actuator US 9 ,856 , 497 B2 59 moiety can be covalently linked ( e . g . , at its N -terminus or least about 2 , 3 , 4 , or 5 hydrophobic residues ) that target a C -terminus ) via a peptide bond to a first portion of the intein protein for export from the cell nucleus to the cytoplasm comprising a C -terminal intein . The actuator moiety - C - through the nuclear pore complex using nuclear transport . terminal intein fusion can be contacted with a second portion Not all NES substrates can be constitutively exported from of the intein sequence comprising an N - terminal intein . This 5 the nucleus . In some embodiments , a targeting sequence contacting of the first and second portion of the inteins can comprises a nuclear localization signal (NLS , e . g . , a SV40 result in a site - specific cleavage ( e . g . , at a suitable site NLS ) and directs a polypeptide to a cell nucleus . A targeting between the actuator moiety and the C -terminal intein ), sequence can direct a polypeptide to a cell nucleus utilizing thereby releasing the actuator moiety . various nuclear localization signals (NLS ) . An NLS can be In some embodiments , the cleavage recognition site com - 10 a monopartite sequence or a bipartite sequence . prises a disulfide bond . The disulfide bond can link the Non - limiting examples of NLSs include and NLS actuator moiety to the chimeric receptor polypeptide . The sequence derived from : the NLS of the SV40 virus large disulfide bond can be formed between one or more cysteines T - antigen , having the amino acid sequence PKKKRKV of the actuator moiety and the receptor . The cysteines can be (SEQ ID NO : 40 ) ; the NLS from nucleoplasmin ( e . g . the engineered into the actuator moiety or receptor. The cyste - 15 nucleoplasmin bipartite NLS with the sequence KRPAAT ines can be a part of the native or wild - type sequence . The KKAGQAKKKK (SEQ ID NO : 41 ) ) ; the c -myc NLS hav cysteines can be present in a linker peptide appended to the ing the amino acid sequence PAAKRVKLD ( SEQ ID NO : actuator moiety or the receptor. Cleavage of the disulfide 42 ) or RQRRNELKRSP (SEQ ID NO : 43 ) ; the hRNPA1 M9 bond can be facilitated by, for example , altering the redox NLS having the sequence NQSSNFGPMKGGNFGGRSS conditions of the disulfide bond . Alteration of the redox 20 GPYGGGGOYFAKPRNOGGY (SEO ID NO : 44 ) ; the conditions can lead to reduction of the disulfide bond to sequence RMRIZFKNKGKDTAELRRRRVEVSVEL thiols and release of the actuator moiety . Cleavage of the RKAKKDEQILKRRNV (SEQ ID NO : 45 ) of the IBB disulfide bond can be facilitated by a cleavage moiety domain from importin -alpha ; the sequences VSRKRPRP comprising a redox agent that can catalyze reduction of the (SEQ ID NO : 46 ) and PPKKARED (SEQ ID NO : 47 ) of the disulfide bond . The redox agent can be an enzyme, or any 25 myoma T protein ; the sequence PQPKKKPL (SEQ ID NO : derivative , variant or fragment thereof. The enzyme can be 48 ) of human ; the sequence SALKKKKKMAP (SEQ ID an oxidoreductase. Examples of include NO : 49 ) ofmouse c - abl IV ; the sequences DRLRR ( SEQ ID protein - disulfide reductase , thioredoxins , glutaredoxins, NO : 50 ) and PKOKKRK ( SEQ ID NO : 51 ) of the influenza thiol disulfide oxidoreductases ( e . g . , DsbA , BdbA - D , virus NS1; the sequence RKLKKKIKKL ( SEQ ID NO : 52 ) MdbA , and SdBA ) , and glutathione disulfide reductase. The 30 of the Hepatitis virus delta antigen ; the sequence REKK redox agent can be from any suitable source including KFLKRR ( SEQ ID NO : 53 ) of the mouse Mx1 protein ; the prokaryotes and eukaryotes . Cofactors ( e . g , nicotinamide sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO : cofactors , flavins, and derivatives and analogs thereof) can 54 ) of the human poly (ADP - ) polymerase ; and the be supplied for optimal activity of the enzyme. sequence RKCLQAGMNLEARKTKK (SEQ ID NO : 55 ) of In an illustrative example shown in FIG . 14A , a chimeric 35 the receptors (human ) glucocorticoid . receptor polypeptide comprises an actuator moiety 1401 In some embodiments , a targeting sequence comprises a linked by disulfide bond . The disulfide bond can be cleaved membrane targeting peptide and directs a polypeptide to a by a cleavage moiety 1402 comprising an enzyme such as an plasma membrane or membrane of a cellular organelle . A oxidoreductase , for example an oxidoreductase complexed membrane -targeting sequence can provide for transport of and / or linked to an adaptor polypeptide as shown in FIG . 40 the chimeric transmembrane receptor polypeptide to a cell 14B . Cleaving of the disulfide bond can release the actuator surface membrane or other cellular membrane . Molecules in moiety as shown in FIG . 14C . The actuator moiety , upon association with cell membranes contain certain regions that release , can translocate to a cell nucleus where it is operable facilitate membrane association , and such regions can be to regulate expression of a target polynucleotide ( e . g . , gene incorporated into a membrane targeting sequence . For expression , and / or activity or edit a nucleic acid sequence as 45 example , some proteins contain sequences at the N - terminus shown in FIG . 14D . FIGS. 14E - H illustrate an alternative or C - terminus that are acylated , and these acyl moieties configuration wherein the actuator moiety is complexed facilitate membrane association . Such sequences can be and / or linked to the adaptor polypeptide and the cleavage recognized by acyltransferases and often conform to a moiety ( e . g . , oxidoreductase ) is linked to the receptor. particular sequence motif . Certain acylation motifs are In some embodiments , the chimeric receptor polypeptide 50 capable of being modified with a single acyl moiety (often comprises at least one targeting sequence which directs followed by several positively charged residues ( e . g . human transport of the receptor to a specific region of a cell . A C -Src ) to improve association with anionic lipid head targeting sequence can be used to direct transport of a groups ) and others are capable of being modified with polypeptide to which the targeting sequence is linked to a multiple acyl moieties . For example the N - terminal specific region of a cell. For example , a targeting sequence 55 sequence of the protein tyrosine kinase Src can comprise a can direct the receptor to a cell nucleus utilizing a nuclear single myristoyl moiety . Dual acylation regions are located localization signal ( NLS ) , outside of the nucleus ( e .g ., the within the N - terminal regions of certain protein kinases, cytoplasm ) utilizing a nuclear export signal (NES ) , the such as a subset of Src family members ( e . g . , Yes, Fyn , Lck ) mitochondria , the endoplasmic reticulum (ER ) , the Golgi, and G -protein alpha subunits . Such dual acylation regions chloroplasts , apoplasts , peroxisomes, plasmamembrane , or 60 often are located within the first eighteen amino acids of membrane of various organelles of a cell . In some embodi - such proteins , and conform to the sequence motif Met -Gly ments , a targeting sequence comprises a nuclear export Cys -Xaa - Cys ( SEQ ID NO : 56 ) , where the Met is cleaved , signal (NES ) and directs a polypeptide outside of a nucleus , the Gly is N -acylated and one of the Cys residues is for example to the cytoplasm of a cell. A targeting sequence S - acylated . The Gly often is myristoylated and a Cys can be can direct a polypeptide to the cytoplasm utilizing various 65 palmitoylated . Acylation regions conforming to the nuclear export signals . Nuclear export signals are generally sequence motif Cys - Ala - Ala -Xaa ( so called " CAAX short amino acid sequences of hydrophobic residues (e . g ., at boxes” ), which can modified with C15 or C10 isoprenyl US 9 ,856 , 497 B2 moieties, from the C - terminus of G -protein gamma subunits sequence . The cell- penetrating domain can be located at the and other proteins also can be utilized . These and other N - terminus, the C - terminus, or anywhere within the actuator acylation motifs include, for example , those discussed in moiety . Gauthier - Campbell et al. , Molecular Biology of the Cell 15 : The targeting sequence can be linked to any appropriate 2205 - 2217 ( 2004 ) ; Glabati et al. , Biochem . J . 303 : 697 - 700 5 region of the chimeric receptor polypeptide , for example at ( 1994 ) and Zlakine et al. , J. Cell Science 110 : 673 -679 the N - terminus, the C -terminus , or in an internal region of ( 1997 ) , and can be incorporated in a targeting sequence to the receptor . In some embodiments , at least two targeting sequences are linked to the receptor. In an exemplary induce membrane localization . chimeric receptor polypeptide shown in FIG . 5 , a first In certain embodiments , a native sequence from a protein1010 targeting sequence 501a can be linked to the extracellular containing an acylation motif is incorporated into a targeting region of the receptor and a second targeting sequence 501b sequence . For example , in some embodiments , an N - termi can be linked to the intracellular region of the receptor, such nal portion of Lck , Fyn or Yes or a G - protein alpha subunit , as to the GMP . When a receptor is linked to multiple such as the first twenty - five N - terminal amino acids or fewer targeting sequences, for example targeting sequences from such proteins ( e . g . , about 5 to about 20 aminomino acids , 15 directed to different locations of a cell, the final localization about 10 to about 19 amino acids, or about 15 to about 19 of the receptor can be determined by the relative strengths of amino acids of the native sequence with optional mutations ), the targeting sequences . For example , a receptor having both may be incorporated within the N - terminus of a chimeric a targeting sequence comprising an NES and a targeting polypeptide . In certain embodiments , a C -terminal sequence sequence comprising an NLS can localize to the cytoplasm of about 25 amino acids or less from a G - protein gamma 20 if the NES is stronger than NLS . Alternatively, if the NLS is subunit containing a CAAX box motif sequence ( e . g ., about stronger than the NES , the receptor can localize to the 5 to about 20 amino acids , about 10 to about 18 amino acids, nucleus even though both a nuclear localization signal and or about 15 to about 18 amino acids of the native sequence nuclear export signal are present on the receptor. A targeting with optional mutations ) can be linked to the C - terminus of sequence can comprise multiple copies of, for example , each a chimeric polypeptide . 25 a NLS and NES , to fine- tune the degree of the cellular Any membrane - targeting sequence can be employed . In localization . In some cases, a targeting sequence is linked to the some embodiments , such sequences include, but are not actuator moiety . Following release of the actuator moiety limited to myristoylation - targeting sequence, palmitoy from the GMP ( and receptor ) by cleavage of the cleavage lation - targeting sequence , prenylation sequences ( i. e ., farneStein 30 recognition site , the targeting sequence can direct the actua sylation , geranyl -geranylation , CAAX Box ) , protein -protein tor moiety to a cellular location that is different from the interaction motifs or transmembrane sequences ( utilizing receptor. For example , a chimeric transmembrane receptor signal peptides ) from receptors . Examples include those can comprise a first targeting sequence directing the receptor discussed in , for example , ten Klooster , J . P . et al, Biology to a plasma membrane and the actuator moiety can sepa of the Cell ( 2007 ) 99 , 1 - 12 ; Vincent, S ., et al ., Naturezatute 35 rately comprise a second targeting sequence directing local Biotechnology 21: 936 -40 , 1098 (2003 ). ization to a cell nucleus . Initially, the actuator moiety Additional protein domains exist that can increase protein (forming a portion of the receptor ) can be localized to a retention at various membranes . For example , an ~ 120 plasma membrane due to the first targeting sequence . Fol amino acid pleckstrin homology (PH ) domain is found in lowing release of the actuator moiety from the GMP by over 200 human proteins that are typically involved in 40 cleavage of the cleavage recognition site , the actuator moi intracellular signaling . PH domains can bind various phos - ety can localize to a cell nucleus via targeting by the second phatidylinositol (PI ) lipids within membranes (e . g . PI ( 3 , 4 , targeting sequence . In some embodiments , the actuator 5 )- P3, PI (3 ,4 ) -P2 , PI ( 4 ,5 )- P2 ) and thus can play a key role moiety translocates to a cell nucleus after cleavage of the in recruiting proteins to different membrane or cellular cleavage recognition sequence . compartments . Often the phosphorylation state of PI lipids 45 Binding of the chimeric adaptor polypeptide to a chimeric is regulated , such as by PI- 3 kinase or PTEN , and thus, receptor polypeptide when the receptor has undergone modi interaction of membranes with PH domains may not be as fication upon binding to an antigen can bring the cleavage stable as by acyl lipids . moiety in proximity to the cleavage recognition site . Cleav In some embodiments , a targeting sequence directing a age of the recognition site can release the actuator moiety polypeptide to a cellular membrane can utilize a membrane 50 from the GMP. Following release , the actuator moiety is anchoring signal sequence . Various membrane- anchoring operable to complex with a target polynucleotide , for sequences are available . For example , membrane anchoring example in the cell cytoplasm or a cell nucleus. Complexing signal sequences of various membrane bound proteins can of the actuator moiety with a target polynucleotide can be used . Sequences can include those from : 1 ) class I regulate the expression and / or activity of at least one gene or integralmembrane proteins such as IL - 2 receptor beta - chain 55 edit a nucleic acid sequence . and insulin receptor beta chain ; 2 ) class II integral mem - In another exemplary configuration , the GMP forms a brane proteins such as neutral endopeptidase ; 3 ) type III portion of the chimeric adaptor polypeptide and the cleavage proteins such as human cytochrome P450 NF25 ; and 4 ) type moiety forms a portion of a chimeric receptor polypeptide . IV proteins such as human P - glycoprotein . A chimeric adaptor polypeptide of an exemplary configura In some embodiments , the chimeric receptor polypeptide 60 tion can comprise ( a ) a receptor binding moiety that binds a is linked to a polypeptide folding domain which can assist in receptor that has undergone modification upon binding to an protein folding . In some embodiments , an actuator moiety is antigen ; and ( b ) a gene modulating polypeptide (GMP ) linked to a cell -penetrating domain . For example, the cell linked to the receptor binding moiety , wherein the GMP penetrating domain can be derived from the HIV - 1 TAT comprises an actuator moiety linked to a cleavage recogni protein , the TLM cell -penetrating motif from human hepa - 65 tion site ; wherein ( i ) the cleavage recognition site is cleav titis B virus , MPG , Pep - 1 , VP22 , a cell penetrating peptide able by a cleavage moiety in response to receptor binding , from Herpes simplex virus, or a polyarginine peptide and ( ii ) the actuator moiety is operable to complex with a US 9 ,856 ,497 B2 63 64 target polynucleotide in response to cleavage of the cleavage protein , or any derivative , variant or fragment thereof , recognition site . FIG . 6A shows an exemplary chimeric selected from focal adhesion kinase ( FAK ) , Src , and protein adaptor polypeptide . A chimeric adaptor polypeptide can phosphatase 2A (PP2A ) . In some embodiments , a chimeric comprise a receptor binding moiety 601 linked to a GMP adaptor polypeptide comprises a protein , or any derivative , 602. AGMP can comprise an actuator moiety 602a linked to 5 variant or fragment thereof , selected from RAB21, PTPN2, a cleavage recognition site 602b . AUP1, BIN1, COL8A1, and ITGB1. A receptor binding moiety of a chimeric adaptor poly - In some embodiments , a chimeric adaptor polypeptide peptide can be any binding partner ( e . g . , protein ) which can comprises a molecule ( e . g . , protein ), or any derivative , bind a receptor, or any derivative , variant or fragment variant or fragment thereof, recruited to a cadherin receptor. thereof. In some embodiments , an adaptor comprises a 10 The molecule may be recruited to the receptor as a result of binding partner of a receptor that is membrane - bound , or any a receptor modification ( e . g ., chemical modification e . g . , derivative , variant or fragment thereof. In some embodi- phosphorylation , and /or conformational change ) . In some ments, an adaptor comprises a binding partner of a receptor, embodiments , a chimeric adaptor polypeptide comprises a or any derivative , variant or fragment thereof, that is not protein , or any derivative , variant or fragment thereof, membrane -bound ( e . g ., intracellular or cytosolic ) . An adap - 15 selected from a - catenin , B - catenin , y - catenin , catenin tor polynucleotide may comprise a receptor binding domain delta - 1 (p120 - catenin ), AJAP1 , CTNND1, DLGAP5 , of a signaling protein or other protein recruited to a receptor. TBC1D2, LIMAI, CAVI , TRPV4 , CTNNB1 complex , The chimeric adaptor polypeptide can be recruited to the PIP5K1C , RAB8B , RAPGEF2, DDR1, PSEN1, CDH1, chimeric receptor polypeptide in response to receptor modi CDC27 , CTNNA1, and EGFR . fication , e .g ., a conformational change , chemical modifica - 20 In some embodiments , a chimeric adaptor polypeptide tion , or combination thereof. A receptor may undergo recep - comprises a molecule ( e . g ., protein ) that is recruited to tor modification in response to ligand binding . Receptors , or chimeric receptor polypeptide comprising a RTSK , or any any derivative , variant or fragment thereof, and binding derivative , variant or fragment thereof. In some embodi partners ( e . g . , proteins ) , or any derivative , variant or frag - ments , a chimeric adaptor polypeptide comprises a protein , ment thereof, can be selected so as to optimize the desired 25 or any derivative , variant or fragment thereof, selected from level of recruitment of the adaptor polypeptide to the recep - a SMAD family member including SMADI, SMAD2 , tor. SMAD3 , SMAD5 , SMAD6 , and SMAD7, and SMAD9 In some embodiments , a chimeric adaptor polypeptide ( sometimes referred to as SMAD8 ) ; the SMAD anchor for comprises a molecule ( e . g . , protein ) , or any derivative , receptor activation (SARA ) ; a SMURF protein ( e . g . , variant or fragment thereof, recruited to a Notch receptor 30 SMURF1, SMURF2 ) ; and any derivative, variant or frag when the Notch receptor is bound to a ligand . A chimeric ment thereof. In some embodiments , a chimeric adaptor adaptor polypeptide can comprise a protein , any derivative , polypeptide comprises a molecule (e .g . , protein ) that is variant or fragment thereof, selected from the group con - recruited to chimeric receptor polypeptide comprising a sisting of presenilin - 1 (PSEN1 ) , nicastrin , anterior pharynx cytokine receptor, or any derivative, variant or fragment defective 1 ( APH - 1 ) , and presenilin enhancer 2 ( PEN - 2 ) . 35 thereof . In some embodiments , an adaptor polypeptide com In some embodiments , a chimeric adaptor polypeptide prises a gp130 , CD131, CD132 , or any derivative , variant or comprises a molecule ( e . g ., protein ) , or any derivative, fragment thereof. variant or fragment thereof, recruited to a GPCR when the In some embodiments , a chimeric adaptor polypeptide GPCR is bound to a ligand ( e . g ., a ligand -bound GPCR that comprises a molecule recruited to a phosphorylated RTK or has undergone conformational and / or biochemical modifi - 40 RTSK , or a receptor phosphorylated by a non - covalently cation ). A chimeric adaptor polypeptide can comprise a associated intracellular kinase . The phosphorylation of spe protein , or any derivative , variant or fragment thereof, cific amino acid residues ( e . g . , tyrosine residues ) within an selected from the group consisting of: AKAP79 (AKAP5 ) activated receptor ( e . g ., a chimeric receptor polypeptide ) can and AKAP250 (AKAP12 , gravin ), arrestin ( e . g ., ( 3 - arres - create binding sites for molecules such as Src homology 2 tin ), ATBP50 , calmodulin , DRIP78 (DNAJC14 ) , Homer , 45 (SH2 ) domain - and phosphotyrosine binding (PTB ) domain GASP1, GEC1 (GABARAPL1 ) , INAD , JAK2, LARG (AR containing proteins. In some embodiments , an adaptor poly HGEF12 ) ,MAGI2 , MAGI3 , M10 MHC , MPP3, MRAP and peptide comprises a protein containing an SH2 domain , such MRAP2 , MUPP1 (MPDZ ) , neurochondrin , NHERF1 as ABL1, ABL2 , BCAR3 , BLK , BLNK , BMX , BTK , ( EBP50 , SLC9A3R1) , NHERF2 (SLC9A3R2 ) , NINAA , CHN2 , CISH , CRK , CRKL , CSK , DAPP1 , EAT - 2 , FER , ODR4, p85 , PDZ -RhoGEF (ARHGEF11 ) , periplakin , PICK 50 FES , FGR , FRK , FYN , GADS , GRAP , GRAP2 , GRB10 , 1 , PSD95 , RACK1 (GNB2L1 ) , RAMP1, RAMP2 , RAMP3 , GRB14 , GRB2, GRB7, HCK , HSH2D , INPP5D , INPPL1, RanBP2 , REEPs , RTPs, RTP4 , Shank , SNX1 , syntrophin , ITK , JAK2 , LCK , LCP2 , LYN , MATK , NCK1, NCK2, spinophilin , TCTEXT1 (DYNLT1 ) , and USP4 . PIK3R1, PIK3R2, PIK3R3 , PLCG1, PLCG2, PTK6 , In some embodiments , a chimeric adaptor polypeptide PTPN11, PTPN6, RASA1 , SAP, SH2B1, SH2B2 , SH2B3 , comprises a molecule ( e . g . , protein ) , or any derivative , 55 SH2D1A , SH2D1B , SH2D2A , SH2D3A , SH2D3C , variant or fragment thereof, that is recruited to an integrin SH2D4A , SH2D4B , SH2D5, SH2D6 , SH3BP2 , SHB , receptor when the receptor is bound to a ligand . Examples SHC1, SHC2, SHC3 , SHC4, SHD , SHE , SHP1, SHP2, of adaptor proteins that are recruited to an integrin receptor SLA , SLA2 , SOCSI , SOCS2 , SOCS3 , SOCS4 , SOCS5 , include , but are not limited to , structural adaptor proteins , SOCS6 , SOCS7, SRC , SRMS , STAT1 , STAT2 , STAT3 , scaffolding adaptor proteins, and adaptor proteins having 60 STAT4 , STAT5A , STAT5B , STAT6 , SUPT6H , SYK , TEC , catalytic activity . In some embodiments , a chimeric adaptor TENC1, TNS , TNS1, TNS3, TNS4 , TXK , VAV1, VAV2 , polypeptide comprises a protein , or any derivative , variant VAV3 , YES1, ZAP70 , or any derivative , variant or fragment or fragment thereof, selected from , kindlin , filamin and thereof. In some embodiments , an adaptor polypeptide com tensin . In some embodiments , a chimeric adaptor polypep - prises a protein containing a PTB domain , such as APBA1, tide comprises a protein , or any derivative , variant or 65 APBA2, APBA3, EPS8 , EPS8L1, EPS8L2, EPS8L3 , fragment thereof, selected from paxillin and kindlin . In some TENCI, TNS , TNS1, TNS3 , TNS4 , DOK1, DOK2 , DOK3 , embodiments , a chimeric adaptor polypeptide comprises a DOK4 , DOK5 , DOKÁ , DOK7, FRS2, FRS3 , IRS1, IRS2, US 9 , 856 , 497 B2 65 66 IRS3 , IRS4 , SHC1, SHC2 , SHC3, SHC4, TLN1, TLN2 , as an engineered ( e. g ., programmable or targetable ) RNA X11a , or any derivative , variant or fragment thereof. nuclease to induce editing of a target RNA sequence. In In some embodiments , a chimeric adaptor polypeptide some embodiments , the actuator moiety has reduced or comprises a protein that is recruited to a TNF receptor , or minimal nuclease activity . An actuator moiety having any derivative , variant or fragment thereof . Such proteins 5 reduced or minimal nuclease activity can regulate expres are sometimes referred to as TNR receptor associated factors sion and /or activity of a gene by physical obstruction of a or TRAFS and include TRAF1 , TRAF2 , TRAF3 , TRAF4 , target polynucleotide or recruitment of additional factors TRAF5, TRAF6 , and TRAF7 . In some embodiments , a effective to suppress or enhance expression of the target chimeric adaptor polypeptide comprises a receptor- interact- polynucleotide . In some embodiments, the actuator moiety ing serine / threonine- 1 (RIP1 or RIPK1) and 10 comprises a nuclease - null DNA binding protein derived receptor - interacting serine / threonine - protein kinase 3 (RIP3 from a DNA nuclease that can induce transcriptional acti or RIPK3 ) , or any derivative , variant or fragment thereof. In vation or repression of a target DNA sequence . In some some embodiments , a chimeric adaptor polypeptide com - embodiments , the actuator moiety comprises a nuclease - null prises an adaptor protein that is recruited to a TNFR , such as RNA binding protein derived from a RNA nuclease that can Fas - associated protein with Dead Domain ( FADD ) and 15 induce transcriptional activation or repression of a target receptor type - 1 associated DEATH RNA sequence . In some embodiments , the actuator moiety domain ( TRADD ) which binds TRAF2 , or any derivative , is a nucleic acid - guided actuator moiety. In some embodi variant or fragment thereof . ments, the actuator moiety is a DNA - guided actuator moiety . In some embodiments , a chimeric adaptor polypeptide In some embodiments , the actuator moiety is an RNA comprises a molecule ( e . g ., protein ) , or any derivative, 20 guided actuator moiety . An actuator moiety can regulate variant or fragment thereof, recruited to a phosphorylated expression or activity of a gene and/ or edit a nucleic acid ITAM , for example an ITAM of a chimeric polypeptide sequence , whether exogenous or endogenous . For example , receptor comprising an immune receptor such as a TCR . The an actuator moiety can comprise a Cas protein which lacks phosphorylation of specific tyrosine residues within the cleavage activity . activated receptor can create binding sites for molecules 25 Any suitable nuclease can be used in an actuator moiety . such as Src homology 2 (SH2 ) domain - and phosphotyrosine Suitable nucleases include , but are not limited to , CRISPR binding (PTB ) domain - containing proteins. In some associated ( Cas ) proteins or Cas nucleases including type I embodiments , a chimeric adaptor polypeptide comprises CRISPR -associated (Cas ) polypeptides, type II CRISPR ABL1, ABL2, BCAR3 , BLK , BLNK , BMX , BTK , CHN2, associated (Cas ) polypeptides, type III CRISPR - associated CISH , CRK , CRKL , CSK , DAPP1, EAT- 2 , FER , FES , FGR , 30 (Cas ) polypeptides, type IV CRISPR - associated (Cas ) poly FRK , FYN , GADS , GRAP, GRAP2 , GRB10 , GRB14 , peptides, type V CRISPR - associated ( Cas ) polypeptides , GRB2, GRB7, HCK , HSH2D , INPP5D , INPPL1, ITK , and type VI CRISPR -associated ( Cas) polypeptides ; zinc JAK2 , LCK , LCP2 , LYN , MATK , NCK1, NCK2 , PIK3R1, finger nucleases (ZFN ) ; transcription activator- like effector PIK3R2, PIK3R3, PLCGI, PLCG2 , PTK6 , PTPN11 , nucleases ( TALEN ) ; meganucleases ; RNA - binding proteins PTPN6 , RASAI , SAP , SH2B1, SH2B2 , SH2B3 , SH2D1A , 35 (RBP ) ; CRISPR - associated RNA binding proteins ; recom SH2D1B , SH2D2A , SH2D3A , SH2D3C , SH2D4A , binases ; flippases ; transposases; Argonaute (Ago ) proteins SH2D4B , SH2D5 , SH2D6 , SH3BP2 , SHB , SHC1, SHC2, ( e . g ., prokaryotic Argonaute (pAgo ) , archaeal Argonaute SHC3, SHC4 , SHD , SHE, SHP1, SHP2 , SLA , SLA2, (aÀgo ) , and eukaryotic Argonaute (eAgo )) ; any derivative SOCSI , SOCS2 , SOCS3 , SOCS4 , SOCS5 , SOCS6 , SOCS7 , thereof, any variant thereof; and any fragment thereof. In SRC , SRMS , STAT1, STAT2 , STAT3 , STAT4 , STAT5A , 40 some embodiments , the actuator moiety comprises a Cas STAT5B , STAT6 , SUPT6H , SYK , TEC , TENCI, TNS , protein that forms a complex with a guide nucleic acid , such TNS1 , TNS3 , TNS4 , TXK , VAV1, VAV2, VAV3, YES1 , as a guide RNA . In some embodiments , the actuator moiety ZAP70 , or any derivative , variant or fragment thereof. In comprises a RNA -binding protein (RBP ) optionally com some embodiments , a chimeric adaptor polypeptide com - plexed with a guide nucleic acid , such as a guide RNA , prises APBA1, APBA2, APBA3, EPS8 , EPS8L1 , EPS8L2 , 45 which is able to form a complex with a Cas protein . EPS8L3 . TENC1, TNS , TNS1, TNS3 , TNS4 , DOK1, In some embodiments , the actuator moiety comprises a DOK2 , DOK3 , DOK4 , DOK5, DOK6 , DOK7, FRS2 , FRS3 , RNA -binding protein (RBP ) optionally complexed with a IRS1 , IRS2 , IRS3 , IRS4 , SHC1, SHC2 , SHC3 , SHC4 , guide nucleic acid , such as a guide RNA , which is able to TLN1, TLN2 , X11a , or any derivative , variant or fragment form a complex with a Cas protein . FIG . 6B shows an thereof. 50 exemplary chimeric adaptor polypeptide in which the actua In some configurations , a chimeric adaptor polypeptide of tor moiety comprises an RNA - binding protein 600a option a subject system can comprise a gene modulating polypep - ally complexed with a guide nucleic acid . Upon release from tide (GMP ). A GMP, as described elsewhere herein , can the RNA -binding protein (RBP ) , for example by dissocia comprise an actuatormoiety linked to a cleavage recognition tion of the guide nucleic acid from the RBP or cleavage of site . The actuator moiety can comprise a nuclease ( e . g ., 55 the cleavage recognition site , the guide nucleic acid can DNA nuclease and / or RNA nuclease ) , modified nuclease form a complex with a Cas protein 600b which is operable ( e . g . , DNA nuclease and/ or RNA nuclease ) that is nuclease to regulate expression of a target polynucleotide ( e . g ., gene deficient or has reduced nuclease activity compared to a expression ) and / or activity or edit a nucleic acid sequence . wild - type nuclease , a variant thereof, a derivative thereof, or In some embodiments , the actuator moiety comprises a a fragment thereof as described elsewhere herein . The 60 nuclease - null DNA binding protein derived from a DNA actuator moiety can regulate expression or activity of a gene nuclease that can induce transcriptional activation or repres and / or edit the sequence of a nucleic acid ( e . g . , a gene and / or sion of a target DNA sequence . In some embodiments , the gene product ). In some embodiments , the actuator moiety actuator moiety comprises a nuclease - null RNA binding comprises a DNA nuclease such as an engineered ( e . g ., protein derived from a RNA nuclease that can induce programmable or targetable ) DNA nuclease to induce 65 transcriptional activation or repression of a target RNA genome editing of a target DNA sequence . In some embodi- sequence . For example , an actuator moiety can comprise a ments , the actuator moiety comprises a RNA nuclease such Cas protein which lacks cleavage activity . US 9 ,856 ,497 B2 67 68 In some embodiments , the cleavage recognition site is following receptor modification ( e . g ., phosphorylation ), the flanked by the receptor binding moiety and the actuator chimeric adaptor polypeptide is recruited to the receptor as moiety . The actuator moiety can be released from the GMP shown in FIG . 10B . A second adaptor polypeptide 1007 and from the chimeric adaptor polypeptide by cleavage of comprising a cleavage moiety 1006 is also recruited to the the recognition site by a cleavage moiety . The cleavage 5 modified receptor ( FIG . 10C ) . The cleavage moiety may be moiety can recognize and /or cleave a cleavage recognition complexed with the second adaptor polypeptide or linked , site , for example , when in proximity to the cleavage recog - for example by a peptide bond and/ or peptide linker, to the nition site . A cleavage moiety can comprise a polypeptide adaptor. When in proximity to the cleavage recognition site , sequence . The cleavage moiety , in some configurations, the cleavage moiety can cleave the recognition site to release forms a portion of the chimeric receptor polypeptide. The 10 the actuator moiety from the GMP as shown in FIG . 10D . cleavage moiety can form the N - terminus , C - terminus or an Upon release , the actuator moiety can enter the nucleus to internal portion of the chimeric receptor polypeptide . In regulate the expression and /or activity of a target gene or some embodiments , the cleavage moiety is complexed to the edit a nucleic acid sequence . FIGS. 10E - H show an analo chimeric receptor polypeptide. The cleavage moiety can be gous system wherein receptor modification comprises a complexed to the N - terminus , C - terminus , or an internal 15 conformational change . In some embodiments , the chimeric portion of the chimeric receptor polypeptide . In an exem - adaptor polypeptide is tethered to the membrane ( e . g . , as a plary configuration shown in FIG . 7 , the cleavage recogni- membrane bound protein ) . In some embodiments , the sec tion site 702b is flanked by the receptor binding moiety 701 ond adaptor polypeptide is tethered to the membrane ( e . g ., and the actuator moiety 702a , and the cleavage moiety 706 as a membrane bound protein ) . forms a portion a chimeric receptor polypeptide 705 . 20 FIGS . 16A - D illustrate schematically the release of an FIGS . 8A - D illustrate schematically the release of an actuator moiety in a system comprising a first membrane actuator moiety from a GMP. FIG . 8A shows the binding of tethered adaptor and a second cytoplasmic adaptor. FIG . an antigen to a transmembrane chimeric receptor polypep - 16A shows the association of a first membrane -tethered tide . The transmembrane chimeric receptor polypeptide adaptor comprising a membrane tethering domain 1601a comprises an extracellular region having an antigen inter - 25 ( e . g . , CAAX ) , a protease recognition site 1601b ( e . g . , TEV ) , acting domain 805 and an intracellular region comprising a and an actuator moiety 1601c with a chimeric transmem cleavage moiety 806 . The cleavage moiety can be com - brane receptor 1602. The chimeric transmembrane receptor plexed with the receptor or linked , for example by a peptide can function as a scaffold and includes at least two adaptor bond and /or peptide linker, to the receptor. The GMP forms binding sites ( e . g . , EGFR or a portion of the chimeric adaptor polypeptide. The GMP, 30 (RTK ) . One adaptor binding site can be associated with a linked to the receptor binding moiety 801 , includes an membrane -tethered adaptor as shown in FIG . 16B . The actuator moiety 802a linked to a cleavage recognition site association of the membrane - tethered adaptor, in some 802b . In response to antigen binding , the receptor is modi- cases , is dependent on antigen binding to the receptor. In fied by phosphorylation 803 in the intracellular region of the some systems, the membrane - tethered adaptor is located in receptor ( FIG . 8B ) . Following receptor modification ( e . g ., 35 proximity to the receptor and association may not depend on phosphorylation ) , the chimeric adaptor polypeptide is antigen binding to the receptor. As shown in FIGS . 16B and recruited to the receptor as shown in FIG . 8C . The receptor 1 6C , antigen interaction with the receptor can conditionally comprises a cleavage moiety 806 . When in proximity to the recruit a second adaptor protein comprising a cytoplasmic cleavage recognition site , the cleavage moiety can cleave the receptor binding moiety 1603a and protease 1603b , to the recognition site to release the actuator moiety from the GMP 40 other adaptor binding site of the receptor. The second as shown in FIG . 8D . Upon release , the actuatormoiety can adaptor protein comprising the protease , when recruited to enter the nucleus to regulate the expression and / or activity of the transmembrane receptor , can cleave the protease recog a target gene or edit a nucleic acid sequence . FIGS. 8E - H nition site 1601b of the membrane -tethered molecule , show an analogous system wherein receptor modification thereby releasing the actuator moiety 1601c as shown in comprises a conformational change. In some embodiments , 45 FIG . 16D . the chimeric adaptor protein is tethered to the membrane In some embodiments , the cleavage moiety only cleaves ( e . g . , as a membrane bound protein ) . at the recognition site when in proximity to the cleavage In another configuration , the cleavage moiety is com - recognition site . In some embodiments , the cleavage recog plexed to a second adaptor polypeptide which binds the nition site comprises a polypeptide sequence ( e . g . , a peptide chimeric receptor polypeptide when the receptor polypep - 50 cleavage domain ) that is a recognition sequence of a pro tide has undergone modification . An illustrative example is tease . The cleavage moiety can comprise protease activity shown in FIG . 9 . The cleavage recognition site 902b is which recognizes the polypeptide sequence . A cleavage flanked by the receptor binding moiety 901 and the actuator moiety comprising protease activity can be a protease moiety 902a , and the cleavage moiety 906 forms a portion including, but not limited to , any protease described else a second adaptor polypeptide 907 . 55 where herein , or any derivative , variant or fragment thereof. FIGS. 10A - D illustrate schematically the release of an In some embodiments , the cleavage recognition site com actuator moiety from a GMP. FIG . 10A shows the binding of prises multiple cleavage recognition sequences, and each an antigen to a transmembrane chimeric receptor polypep - cleavage recognition sequence can be recognized by the tide . The transmembrane chimeric receptor polypeptide same or different cleavage moiety comprising protease comprises an extracellular region having an antigen inter - 60 activity ( e . g . , protease ) . acting domain and an intracellular region . The GMP, com - In some embodiments , the cleavage recognition site com prising an actuator moiety linked to a cleavage recognition prises a first portion of an intein sequence that reacts with the site , forms a portion of a chimeric adaptor polypeptide. The second portion of the intein sequence to release the actuator cleavage recognition site 1002b is flanked by the receptor moiety . A heterologous split intein system can be used to binding moiety 1001 and the actuator moiety 1002a . In 65 facilitate release of the actuator moiety from the chimeric response to antigen binding , the receptor is modified by adaptor polypeptide . The actuator moiety can be covalently phosphorylation 1003 in the intracellular region (FIG . 10B ). linked to the first portion of the intein sequence . The actuator US 9 ,856 , 497 B2 69 70 moiety can be linked via its N - terminus or C - terminus to the some embodiments , a targeting sequence comprises a first portion of the intein sequence . The cleavage moiety can nuclear localization signal (NLS ) and directs the adaptor to comprise the second portion of the intein sequence . The first a cell nucleus . A targeting sequence can direct the adaptor to portion or second portion of the intein sequence can be the a cell nucleus utilizing various nuclear localization signals N - terminal intein , the C - terminal intein , or any other suit - 5 (NLS ) . In some embodiments , a targeting sequence com able portion of an intein that can facilitate release of the prises a membrane targeting sequence and directs the adap actuator moiety . The intein sequences can be from any tor to a plasma membrane or membrane of a cellular suitable source . The first and second portion can be from the organelle . A targeting sequence can direct a polypeptide to same or different sources ( e . g ., organism , protein ) . In an a membrane utilizing a membrane anchoring signal illustrative example , an actuator moiety can be covalently 10 sequence as previously described . Various membrane - an linked ( e . g ., at its N - terminus or C - terminus ) via a peptide choring sequences are available . bond to a first portion of the intein sequence, which com The targeting sequence can be linked to any appropriate prises an N - terminal intein . The actuator moiety - N - terminal region of the chimeric adaptor polypeptide, for example at intein fusion can be contacted with a second portion of the the N -terminus or the C -terminus of the polypeptide or in an intein sequence comprising a C -terminal intein . This con - 15 internal region of the adaptor. In some embodiments , at least tacting of the first and second portion of the intein sequences two targeting sequences are linked to the adaptor. For can result in a site specific cleavage ( e . g . , at a site between example , as shown in FIG . 11 , a first targeting sequence the actuator moiety and the N - terminal intein ) , thereby 1101a can be linked to the receptor binding moiety of the releasing the actuator moiety . In another illustrative adaptor and a second targeting sequence 1101b can be linked example , an actuator moiety can be covalently linked ( e . g ., 20 to the GMP of the adaptor, for example to the actuator at its N -terminus or C - terminus ) via a peptide bond to a first moiety . When an adaptor is linked to multiple targeting portion of the intein comprising a C -terminal intein . The sequences, for example targeting sequences directed to actuator moiety - C - terminal intein fusion can be contacted different locations of a cell , the final localization of the with a second portion of the intein sequence comprising an adaptor can be determined by the relative strengths of the N - terminal intein . This contacting of the first and second 25 targeting sequences . For example , an adaptor having both a portion of the inteins can result in a site -specific cleavage targeting sequence comprising an NES and a targeting ( e . g ., at a suitable site between the actuator moiety and the sequence comprising an NLS can localize to the cytosol if C - terminal intein ) , thereby releasing the actuator moiety . the NES is stronger than the NLS . Alternatively , if the NLS In some embodiments , the cleavage recognition site com - is stronger than the NES , the adaptor can localize to the prises a disulfide bond . The disulfide bond can link the 30 nucleus even though both a nuclear localization signal and actuator moiety to the receptor binding moiety in a chimeric nuclear export signal are present on the adaptor . A targeting adaptor polypeptide. The disulfide bond can be formed sequence can comprise multiple copies of, for example , each between one or more cysteines of the actuator moiety and a NLS and NES , to fine -tune the degree of the cellular the receptor binding moiety . The cysteines can be engi - localization . neered into the actuator moiety or receptor binding moiety. 35 In some cases, a targeting sequence is linked to the The cysteines can be a part of the native or wild -type actuator moiety . Following release of the actuator moiety sequence of the actuator moiety or receptor binding moiety . from the GMP (and adaptor ) by cleavage of the cleavage The cysteines can be present in a linker peptide appended to recognition site , the targeting sequence can direct the actua the actuator moiety or the receptor binding moiety . Cleavage tor moiety to a cellular location that is different from the of the disulfide bond can be facilitated by, for example , 40 adaptor. For example , a chimeric adaptor polypeptide can altering the redox conditions of the disulfide bond . Altera comprise a first targeting sequence directing the adaptor to tion of the redox conditions can lead to reduction of the the cell cytoplasm and the actuator moiety can separately disulfide bond to thiols and release of the actuator moiety. comprise a second targeting sequence directing localization Cleavage of the disulfide bond can be facilitated by a to a cell nucleus . Initially , the actuator moiety ( forming a cleavage moiety comprising a redox agent that can lead to 45 portion of the adaptor ) can be localized to the cell cytoplasm reduction of the disulfide bond . The redox agent can be an due to the first targeting sequence . Following release of the enzyme, or any derivative , variant or fragment thereof . The actuator moiety from the GMP by cleavage of the cleavage enzyme can be an oxidoreductase . Examples of oxidoreduc - recognition site , the actuator moiety can localize to a cell tases include protein - disulfide reductase , thioredoxins, glu - nucleus via targeting by the second targeting sequence . In taredoxins , thiol disulfide Oxidoreductases ( e . g . , DsbA , 50 some embodiments , the actuator moiety translocates to a cell BdbA - D , MdbA , SdbA ) , and glutathione disulfide reduc nucleus after cleavage of the cleavage recognition sequence . tase. The redox agent can be from any suitable source In some embodiments , a targeting sequence comprises a including prokaryotes and eukaryotes . Cofactors (e . g , nico - membrane targeting peptide and directs a polypeptide to a tinamide cofactors, flavins , and derivatives and analogs plasma membrane or membrane of a cellular organelle . A thereof) can be supplied for optimal activity of the enzyme . 55 membrane -targeting sequence can provide for transport of In some embodiments , the chimeric adaptor polypeptide the chimeric transmembrane receptor polypeptide to a cell comprises at least one targeting sequence which directs surface membrane or other cellular membrane. Any suitable transport of the adaptor to a specific region of a cell . For membrane target sequence previously described herein may example , a targeting sequence can direct the adaptor to a cell be used . nucleus utilizing a nuclear localization signal (NLS ) , outside 60 In some embodiments , the chimeric adaptor polypeptide of a cell nucleus (e . g. , to the cytoplasm ) utilizing a nuclear is linked to a polypeptide folding domain which can assist in export signal (NES ) , the mitochondria , the endoplasmic protein folding. In some embodiments , an actuator moiety reticulum (ER ) , the Golgi, chloroplasts , apoplasts , peroxi can be linked to a cell -penetrating domain . For example , the somes, plasma membrane , or membrane of various organ - cell - penetrating domain can be derived from the HIV - 1 TAT elles of a cell. In some embodiments, a targeting sequence 65 protein , the TLM cell- penetrating motif from human hepa comprises a nuclear export signal (NES ) and directs the titis B virus , MPG , Pep - 1 , VP22 , a cell penetrating peptide chimeric adaptor polypeptide outside of a cell nucleus. In from Herpes simplex virus, or a polyarginine peptide US 9 ,856 ,497 B2 71 72 sequence . The cell -penetrating domain can be located at the A chimeric intracellular receptor comprising a nuclear N - terminus , the C -terminus , or anywhere within the actuator receptor, or any derivative , variant or fragment thereof, can moiety . bind an antigen comprising any suitable ligand of a nuclear The actuator moiety of a subject system , upon release receptor, or any derivative , variant or fragment thereof. from a chimeric adaptor polypeptide or chimeric receptor 5 Non -limiting examples of ligands of nuclear receptors polypeptide , can bind to a target polynucleotide to regulate include thyroid hormone , vitamin A and related compounds , expression and/ or activity of the target polynucleotide by fatty acids, , heme, cholesterol, ATRA , Oxys physical obstruction of the target polynucleotide or recruit - terols , vitamin D , xenobiotics , androstane , , estro ment of additional factors effective to suppress or enhance gens , cortisol, aldosterone , , , and expression of the target polynucleotide . In some embodi- 10 phosphatidylinositols . In some embodiments , the antigen is ments , the actuator moiety comprises a transcriptional acti a hormone . vator effective to increase expression of the target polynucle FIGS . 12A - C illustrates schematically a system compris otide . The actuator moiety can comprise a transcriptional ing an exemplary intracellular receptor comprising a nuclear repressor effective to decrease expression of the target receptor. The system includes a receptor 1200 comprising an polynucleotide . In some embodiments, the target polynucle - 15 actuator moiety 1201 . In the absence of a ligand binding to otide comprises genomic DNA . In some embodiments, the the nuclear receptor, the receptor can be sequestered in a target polynucleotide comprises a region of a plasmid , for certain compartment of a cell , for example the cytoplasm , by example a plasmid carrying an exogenous gene . In some interaction with a binding protein 1202 as shown in FIG . embodiments , the target polynucleotide comprises RNA , for 12A . Upon binding of a ligand 1203 to the intracellular example mRNA . In some embodiments , the target poly - 20 receptor as shown in FIG . 12B , the receptor can dissociate nucleotide comprises an endogenous gene or gene product . from the binding protein 1202 and translocate to the nucleus . The actuator moiety can include one or more copies of a The actuator moiety 1201 which is complexed and / or linked nuclear localization signal that allows the actuator to trans - to the receptor enters the nucleus with the receptor where it locate into the nucleus upon cleavage from the GMP. is operable to regulate expression of a target polynucleotide In some aspects , the chimeric receptor polypeptide is a 25 ( e . g ., gene expression ) and/ or activity or edit a nucleic acid chimeric intracellular receptor. An exemplary chimeric sequence . intracellular receptor comprises ( a ) an antigen interacting The actuator moiety can comprise a nuclease ( e . g ., DNA domain that specifically binds an antigen , and ( b ) an actuator nuclease and /or RNA nuclease ) , modified nuclease ( e . g . , moiety linked to the antigen interacting domain . In some DNA nuclease and / or RNA nuclease ) that is nuclease embodiments , (i ) the chimeric intracellular receptor is modi- 30 deficient or has reduced nuclease activity compared to a fied in response to antigen binding, ( ii ) the chimeric intra - wild - type nuclease , a variant thereof , a derivative thereof , or cellular receptor polypeptide translocates to a nucleus of a a fragment thereof as described elsewhere herein . The cell in response to modification , and (iii ) the actuator moiety actuator moiety can regulate expression or activity of a gene complexes with a target polynucleotide in the nucleus . and /or edit the sequence of a nucleic acid ( e . g ., a gene and /or In some embodiments , a chimeric intracellular receptor is 35 gene product) . In some embodiments , the actuator moiety a nuclear receptor. For example , a chimeric intracellular comprises a DNA nuclease such as an engineered ( e . g . , receptor polypeptide can comprise a nuclear receptor, or any programmable or targetable ) DNA nuclease to induce derivative , variant or fragment thereof, selected from a genome editing of a target DNA sequence . In some embodi a ( TRa ), thyroid hormone recep - ments, the actuator moiety comprises a RNA nuclease such tor B ( TRB ) , - a (RAR - a ) , retinoic acid 40 as an engineered ( e . g . , programmable or targetable ) RNA receptor- B (RAR - B ) , retinoic acid receptor- y (RAR - Y ) , per - nuclease to induce editing of a target RNA sequence . In oxisome proliferator- activated receptor - a ( PPARa ) , peroxi- some embodiments, the actuator moiety has reduced or some proliferator -activated receptor - B / 8 (PPAR - B / 8 ), per- minimal nuclease activity . An actuator moiety having oxisome proliferator- activated receptor - y (PPARY ) , Rev reduced or minimal nuclease activity can regulate expres ErbAa , Rev - ErbAB , RAR - related - a 45 sion and / or activity of a gene by physical obstruction of a (RORa ) , RAR - related orphan receptor- ß (RORB ) , RAR - target polynucleotide or recruitment of additional factors related orphan receptor -y (RORY ) , - a , Liver effective to suppress or enhance expression of the target X receptor - B , , Farnesoid X receptor - B , polynucleotide . In some embodiments , the actuator moiety , , constitutive adros - comprises a nuclease -null DNA binding protein derived tane receptor, hepatocyte nuclear factor- 4 - a (HNF4a ), 50 from a DNA nuclease that can induce transcriptional acti hepatocyte nuclear factor- 4 - a (HNF4y ), X recep - vation or repression of a target DNA sequence . In some tor- a (RXRa ) , - ß (RXRB ) , retinoid X embodiments , the actuator moiety comprises a nuclease - null receptor- y (RXRy ) , 2 (TR2 ), testicular RNA binding protein derived from a RNA nuclease that can receptor 4 ( TR4 ), homologue of the Drosophila tailless gene i nduce transcriptional activation or repression of a target ( TLX ) , photoreceptor cell - specific nuclear receptor ( PNR ) , 55 RNA sequence . In some embodiments , the actuator moiety ovalbumin upstream promoter- transcription factor I is a nucleic acid - guided actuator moiety . In some embodi (COUP - TFI) , chicken ovalbumin upstream promoter- tran - ments , the actuator moiety is a DNA - guided actuator moiety . scription factor II ( COUP - TFII ), V -erbA - related (EAR - 2 ), In some embodiments , the actuator moiety is an RNA estrogen receptor- a ( ERa ), estrogen receptor - B (ERB ) , guided actuator moiety . An actuator moiety can regulate estrogen -related receptor- a ( ERRa ) , estrogen - related recep - 60 expression or activity of a gene and / or edit a nucleic acid tor- ß ( ERRB ), estrogen -related receptor - y ( ERRY ), glucocor - sequence , whether exogenous or endogenous. ticoid receptor (GR ) , mineralocorticoid receptor (MR ) , pro - Any suitable nuclease can be used in an actuator moiety . gesterone receptor (PR ), (AR ), nerve Suitable nucleases include , but are not limited to , CRISPR growth factor IB (NGFIB ) , nuclear receptor related i associated (Cas ) proteins or Cas nucleases including type I (NURR1 ) , neuron -derived orphan receptor 1 (NOR1 ) , ste - 65 CRISPR - associated (Cas ) polypeptides , type II CRISPR roidogenic factor 1 ( SF1 ) , liver receptor homolog - 1 (LRH associated (Cas ) polypeptides , type III CRISPR -associated 1 ) , and (GCNF ) . (Cas ) polypeptides , type IV CRISPR - associated (Cas ) poly US 9 ,856 , 497 B2 73 74 peptides, type V CRISPR - associated ( Cas ) polypeptides, A cell can be a stem cell or progenitor cell. Cells can and type VI CRISPR - associated ( Cas) polypeptides; zinc include stem cells ( e . g ., adult stem cells, embryonic stem finger nucleases (ZFN ) ; transcription activator - like effector cells , iPS cells ) and progenitor cells ( e . g . , cardiac progenitor nucleases ( TALEN ) ; meganucleases; RNA - binding proteins cells , neural progenitor cells , etc . ) . Cells can include mam (RBP ) ; CRISPR - associated RNA binding proteins ; recom - 5 malian stem cells and progenitor cells , including rodent stem binases; flippases; transposases ; Argonaute proteins ( e. g ., cells , rodent progenitor cells , human stem cells , human prokaryotic Argonaute ( Ago ) , archaeal Argonaute ( aAgo ) , progenitor cells, etc . Clonal cells can comprise the progeny and eukaryotic Argonaute ( eAgo ) ) ; any derivative thereof of a cell . A cell can comprise a target nucleic acid . A cell can any variant thereof and any fragment thereof. In some be in a living organism . A cell can be a genetically modified embodiments , the actuator moiety comprises a Cas protein 10 cell . A cell can be a host cell . that forms a complex with a guide nucleic acid , such as a A cell can be a totipotent stem cell , however , in some guide RNA . embodiments of this disclosure , the term “ cell ” may be used The actuator moiety of an intracellular receptor , upon but may not refer to a totipotent stem cell . A cell can be a translocation to a cell nucleus ( e .g . , with the intracellular plant cell , but in some embodiments of this disclosure , the receptor ) , can bind to a target polynucleotide to regulate 15 term “ cell” may be used but may not refer to a plant cell. A expression and / or activity of the target polynucleotide by cell can be a pluripotent cell . For example , a cell can be a physical obstruction of the target polynucleotide or recruit - pluripotent hematopoietic cell that can differentiate into ment of additional factors effective to suppress or enhance other cells in the hematopoietic cell lineage but may not be expression of the target polynucleotide . In some embodi- able to differentiate into any other non - hematopoetic cell. A ments, the actuator moiety comprises a transcriptional acti - 20 cell may be able to develop into a whole organism . A cell vator effective to increase expression of the target polynucle may or may not be able to develop into a whole organism . otide . The actuator moiety can comprise a transcriptional A cell may be a whole organism . repressor effective to decrease expression of the target A cell can be a primary cell. For example , cultures of polynucleotide. primary cells can be passaged 0 times, 1 time, 2 times , 4 In some embodiments , the target polynucleotide com - 25 times , 5 times, 10 times, 15 times or more . Cells can be prises genomic DNA . In some embodiments , the target unicellular organisms. Cells can be grown in culture . polynucleotide comprises a region of a plasmid , for example A cell can be a diseased cell. A diseased cell can have a plasmid carrying an exogenous gene . In some embodi- altered metabolic , gene expression , and /or morphologic fea ments , the target polynucleotide comprises RNA , for tures. A diseased cell can be a cancer cell , a diabetic cell , and example mRNA . In some embodiments , the target poly - 30 a apoptotic cell . A diseased cell can be a cell from a diseased nucleotide comprises an endogenous gene or gene product. subject. Exemplary diseases can include blood disorders , In some cases, a targeting sequence is linked to the cancers ,metabolic disorders, eye disorders, organ disorders, intracellular receptor. For example , a targeting sequence can musculoskeletal disorders , cardiac disease , and the like . direct the receptor to a cell nucleus utilizing a nuclear I f the cells are primary cells , they may be harvested from localization signal (NLS ) , outside of a cell nucleus ( e . g . , to 35 an individual by any method . For example , leukocytes may the cytoplasm ) utilizing a nuclear export signal (NES ) , the be harvested by apheresis , leukocytapheresis , density gra mitochondria , the endoplasmic reticulum ( ER ), the Golgi, dient separation , etc . Cells from tissues such as skin , muscle , chloroplasts , apoplasts, or peroxisomes . In some embodi- bone marrow , spleen , liver, , lung , intestine , stom ments , a targeting sequence comprises a nuclear export a ch , etc . can be harvested by biopsy. An appropriate solution signal (NES ) and directs the receptor outside of a cell 40 may be used for dispersion or suspension of the harvested nucleus. In some embodiments, a targeting sequence com - cells. Such solution can generally be a balanced salt solu prises a nuclear localization signal (NLS ) and directs the tion , ( e . g . normal saline, phosphate -buffered saline ( PBS) , receptor to a cell nucleus . A targeting sequence can direct the Hank ' s balanced salt solution , etc . ) , conveniently supple receptor to a cell nucleus utilizing various nuclear localiza - mented with fetal calf serum or other naturally occurring tion signals (NLS ) . In some embodiments , the chimeric 45 factors , in conjunction with an acceptable buffer at low intracellular receptor is linked to a polypeptide folding concentration . Buffers can include HEPES , phosphate buf domain which can assist in protein folding . fers , lactate buffers , etc . Cells may be used immediately , or A subject system can be introduced into a variety of cells . they may be stored ( e . g . , by freezing ) . Frozen cells can be A cell can be in vitro . A cell can be in vivo . A cell can be ex thawed and can be capable of being reused . Cells can be vivo . A cell can be an isolated cell . A cell can be a cell inside 50 frozen in a DMSO , serum , medium buffer ( e . g ., 10 % of an organism . A cell can be an organism . A cell can be a DMSO , 50 % serum , 40 % buffered medium ) , and / or some cell in a cell culture . A cell can be one of a collection of cells . other such common solution used to preserve cells at freez A cell can be a mammalian cell or derived from a mamma ing temperatures . lian cell. A cell can be a rodent cell or derived from a rodent Non - limiting examples of cells in which a subject system cell. A cell can be a human cell or derived from a human cell . 55 can be utilized include , but are not limited to , lymphoid A cell can be a prokaryotic cell or derived from a prokaryotic cells , such as B cell , T cell (Cytotoxic T cell, . A cell can be a bacterial cell or can be derived from a T cell, Regulatory T cell , ), Natural killer cell , bacterial cell. A cell can be an archaeal cell or derived from cytokine induced killer (CIK ) cells (see e . g . an archaeal cell. A cell can be a eukaryotic cell or derived US20080241194 ) ; myeloid cells , such as granulocytes (Ba from a eukaryotic cell. A cell can be a pluripotent stem cell. 60 sophil granulocyte , Eosinophil granulocyte , Neutrophil A cell can be a plant cell or derived from a plant cell. A cell granulocyte /Hypersegmented neutrophil ), Monocyte /Mac can be an animal cell or derived from an animal cell . A cell rophage , (Reticulocyte ) , Mast cell, Throm can be an invertebrate cell or derived from an invertebrate bocyte /Megakaryocyte , ; cells from the endo cell. A cell can be a vertebrate cell or derived from a crine system , including thyroid ( Thyroid epithelial cell , vertebrate cell. A cell can be a microbe cell or derived from 65 Parafollicular cell) , parathyroid (Parathyroid chief cell , a microbe cell . A cell can be a fungi cell or derived from a Oxyphil cell ) , adrenal (Chromaffin cell ) , pineal ( Pinealo fungi cell. A cell can be from a specific organ or tissue . cyte ) cells ; cells of the , including glial cells US 9 ,856 ,497 B2 75 76 ( , Microglia ), Magnocellular neurosecretory cell, dig cell of testes , Theca interna cell of ovarian follicle , Stellate cell, Boettcher cell , and pituitary (Gonadotrope , Corpus luteum cell of ruptured ovarian follicle , Granulosa Corticotrope , Thyrotrope , Somatotrope, Lactotroph ) ; cells lutein cells , Theca lutein cells , Juxtaglomerular cell ( renin of the Respiratory system , including Pneumocyte ( Type I secretion ), Macula densa cell of kidney , Metabolism and pneumocyte , Type II pneumocyte ) , Clara cell , Goblet cell , 5 storage cells , Barrier function cells (Lung , Gut, Exocrine Dust cell ; cells of the circulatory system , including Myo - Glands and Urogenital Tract) , Kidney , Type I pneumocyte cardiocyte , Pericyte ; cells of the digestive system , including ( lining air space of lung ), Pancreatic duct cell ( centroacinar stomach (Gastric chief cell , Parietal cell ) , Goblet cell, cell) , Nonstriated duct cell (of sweat gland , salivary gland , Paneth cell, G cells , D cells , ECL cells , I cells , K cells , S , etc . ) , Duct cell (of seminal vesicle , prostate cells ; enteroendocrine cells , including enterochromaffin cell, 10 gland , etc . ) , Epithelial cells lining closed internal body APUD cell , liver (Hepatocyte , Kupffer cell ), Cartilage /bone ) cavities, Ciliated cells with propulsive function , Extracellu muscle ; bone cells , including Osteoblast, Osteocyte , Osteo - lar matrix secretion cells , Contractile cells ; Skeletal muscle clast , teeth (Cementoblast , Ameloblast ) ; cartilage cells , cells , stem cell, Heart muscle cells , Blood and immune including Chondroblast , Chondrocyte ; skin cells , including system cells , Erythrocyte (red blood cell ), Megakaryocyte Trichocyte , , Melanocyte (Nevus cell) ; muscle 15 (platelet precursor ), Monocyte , Connective tissue macro cells , including Myocyte ; urinary system cells , including phage ( various types ) , Epidermal Langerhans cell , Osteo Podocyte , Juxtaglomerular cell, Intraglomerular mesangial clast ( in bone ), Dendritic cell (in lymphoid tissues ) ,Micro cell/ Extraglomerular mesangial cell , Kidney proximal glial cell (in ), Neutrophil tubule brush border cell , Macula densa cell; reproductive granulocyte , Eosinophil granulocyte , Basophil granulocyte , system cells , including Spermatozoon , Sertoli cell , Leydig 20 Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell , cell, Ovum ; and other cells, including Adipocyte , Fibroblast, Natural Killer T cell , B cell , Natural killer cell, Reticulocyte , Tendon cell, Epidermal keratinocyte ( differentiating epider - Stem cells and committed progenitors for the blood and mal cell) , Epidermal basal cell (stem cell ), Keratinocyte of immune system (various types ) , Pluripotent stem cells , fingernails and toenails , Nail bed basal cell ( stem cell ) , Totipotent stem cells , Induced pluripotent stem cells , adult Medullary hair shaft cell, Cortical hair shaft cell, Cuticular 25 stem cells , Sensory transducer cells , Autonomic neuron hair shaft cell , Cuticular hair root sheath cell , Hair root cells , Sense organ and peripheral neuron supporting cells , sheath cell of Huxley ' s layer, Hair root sheath cell of Central nervous system neurons and glial cells , Lens cells , Henle ' s layer , External hair root sheath cell , Hair matrix cell Pigment cells , Melanocyte , Retinal pigmented epithelial ( stem cell ) , Wet stratified barrier epithelial cells , Surface cell , Germ cells , Oogonium /Oocyte , Spermatid , Spermato epithelial cell of stratified squamous epithelium of cornea , 30 cyte , Spermatogonium cell (stem cell for spermatocyte ) , tongue , oral cavity, esophagus , anal canal, distal urethra and Spermatozoon , Nurse cells , Ovarian follicle cell, Sertoli cell vagina , basal cell ( stem cell) of epithelia of cornea , tongue , (in testis ) , epithelial cell , Interstitial cells , and oral cavity , esophagus, anal canal, distal urethra and vagina , Interstitial kidney cells . Urinary epithelium cell ( lining urinary bladder and urinary A subject system introduced into a cell can be used for ducts ) , Exocrine secretory epithelial cells , Salivary gland 35 regulating expression of a target polynucleotide ( e . g . , gene mucous cell (polysaccharide -rich secretion ), Salivary gland expression ) . In an aspect, the disclosure provides methods of serous cell ( glycoprotein enzyme- rich secretion ), Von regulating expression of a target polynucleotide in a cell . In Ebner ' s gland cell in tongue (washes taste buds ) ,Mammary some embodiments , the method comprises ( a ) exposing a gland cell (milk secretion ) , Lacrimal gland cell ( tear secre chimeric receptor polypeptide to an antigen , wherein (i ) the tion ) , Ceruminous gland cell in ear (wax secretion ) , Eccrine 40 chimeric receptor polypeptide is modified upon exposure to sweat gland dark cell ( glycoprotein secretion ), Eccrine the antigen , and ( ii ) receptor modification comprises a sweat gland clear cell ( small molecule secretion ) . Apocrine conformational change or a chemical modification ; ( b ) bind sweat gland cell ( odoriferous secretion , sex -hormone sensi- ing a chimeric adaptor polypeptide to the receptor in tive ) , Gland of Moll cell in eyelid ( specialized sweat gland) , response to the modification to form a complex between a cell ( lipid - rich sebum secretion ) , Bow - 45 gene modulating polypeptide (GMP ) and a cleavage moiety , man ' s gland cell in nose (washes olfactory epithelium ), wherein the GMP comprises an actuator moiety linked to a Brunner' s gland cell in duodenum ( enzymes and alkaline cleavage recognition site ; and (c ) cleaving the cleavage mucus ), Seminal vesicle cell (secretes seminal fluid com - recognition site with the cleavage moiety , wherein upon ponents , including fructose for swimming sperm ), Prostate cleavage of the cleavage recognition site , the actuator moi gland cell ( secretes seminal fluid components ) , Bulboure - 50 ety is activated to complex with a target polynucleotide . In thral gland cell (mucus secretion ), Bartholin ' s gland cell some embodiments, the GMP forms a portion of an intrac ( vaginal lubricant secretion ), Gland of Littre cell (mucus ellular region of the chimeric receptor polypeptide, and the secretion ) , cell ( carbohydrate secre - cleavage moiety forms part of the chimeric adaptor poly tion ), Isolated goblet cell of respiratory and digestive tracts peptide. In some embodiments , the GMP forms a portion of (mucus secretion ) , Stomach lining mucous cell (mucus 55 the chimeric adaptor polypeptide, and the cleavage moiety secretion ) , Gastric gland zymogenic cell (pepsinogen secre forms a portion of an intracellular portion of the chimeric tion ) , Gastric gland oxyntic cell ( hydrochloric acid secre - receptor polypeptide . In some embodiments , the cleavage tion ) , Pancreatic acinar cell (bicarbonate and digestive moiety is complexed with a second adaptor polypeptide that enzyme secretion ) , Paneth cell of small intestine ( lysozyme binds the receptor in response to the receptor modification , secretion ), Type II pneumocyte of lung (surfactant secre - 60 and the GMP forms a portion of the chimeric adaptor tion ) , Clara cell of lung , Hormone secreting cells , Anterior polypeptide . pituitary cells , Somatotropes , Lactotropes , Thyrotropes, chimeric receptor polypeptide can be any chimeric Gonadotropes , Corticotropes, Intermediate pituitary cell , receptor polypeptide described herein . In some embodi Magnocellular neurosecretory cells , Gut and respiratory ments, the chimeric receptor polypeptide is a transmem tract cells , Thyroid gland cells , thyroid epithelial cell , para - 65 brane receptor. For example, a chimeric transmembrane follicular cell, Parathyroid gland cells , Parathyroid chief receptor polypeptide comprises a G -protein coupled recep cell , Oxyphil cell, Adrenal gland cells , chromaffin cells , Ley t or (GPCR ) such as Wnt receptor (e . g. , Frizzled family US 9 ,856 ,497 B2 77 78 receptors ) ; integrin receptor ; cadherin receptor, catalytic residue of a chimeric receptor polypeptide. Binding of a receptor including receptors possessing enzymatic activity chimeric adaptor polypeptide to the chimeric receptor poly and receptors which , rather than possessing intrinsic enzy - peptide in response to receptor modification can form a matic activity , act by stimulating non - covalently associated complex between a GMP and a cleavage moiety . Formation enzymes ( e . g . , kinases ) ; death receptor such as members of 5 of a complex between the GMP and cleavage moiety can the tumor necrosis factor receptor superfamily ; immune result in cleavage of the cleavage recognition site by the receptor such as T - cell receptors ; or any derivative , variant, cleavage moiety . In some embodiments , the cleavage rec or fragment thereof. In some embodiments , the receptor ognition site comprises a polypeptide sequence ( e . g . , a does not comprise SEQ ID NO : 39 . peptide cleavage domain ) recognized by a cleavage moiety Exposing a chimeric receptor polypeptide expressed in a 10 comprising protease activity . The cleavage moiety can com cell to an antigen can be conducted in vitro and/ or in vivo . prise protease activity which recognizes the polypeptide Exposing a chimeric receptor polypeptide expressed in a cell sequence . A cleavage moiety comprising protease activity to an antigen can comprise to bringing the receptor in can be a protease including , but not limited , to any protease contact with the antigen , which can be a membrane -bound described elsewhere herein , or any derivative , variant or antigen or non -membrane bound antigen . The antigen is , in 15 fragment thereof. In some embodiments , the cleavage rec some cases , bound the membrane of a cell . The antigen is , ognition site comprises multiple cleavage recognition in some cases , not bound the membrane of a cell . Exposing sequences , and each cleavage recognition sequence can be a cell to an antigen can be conducted in vitro by culturing the recognized by the same or different cleavage moiety com cell expressing a subject system in the presence of the prising protease activity ( e .g ., protease ) . In some embodi antigen . For example , a cell expressing subject system can 20 ments , receptor modification comprises modification atmul be cultured as an adherent cell or in suspension , and the tiple modification sites, and each modification is effective to antigen can be added to the cell culture media . In some bind a chimeric adaptor polypeptide . In some embodiments , cases, the antigen is expressed by a target cell , and exposing (i ) the GMP forms a portion of the chimeric adaptor poly can comprise co -culturing the cell expressing a subject peptide, ( ii ) the chimeric adaptor polypeptide is released system and the target cell expressing the antigen . Cells can 25 from the chimeric receptor polypeptide following cleavage be co -cultured in various suitable types of cell culture media , of the cleavage recognition site , and ( iii ) a further chimeric for example with supplements , growth factors , ions, etc . adaptor polypeptide comprising a GMP binds the modified Exposing a cell expressing a subject system to a target cell receptor. ( e . g . , a target cell expressing an antigen ) can be accom - In some embodiments , the cleavage recognition site com plished in vivo , in some cases , by administering the cells to 30 prises a first portion of an intein sequence that reacts with the a subject, for example a human subject, and allowing the second portion of the intein sequence to release the actuator cells to localize to the target cell via the circulatory system . moiety . A heterologous split intein system , as described Exposing can be performed for any suitable length of elsewhere herein , can be used to facilitate release of the time, for example at least 1 minute , at least 5 minutes , at actuator moiety . The actuator moiety can be covalently least 10 minutes, at least 30 minutes , at least 1 hour, at least 35 linked to the first portion of the intein sequence . The actuator 2 hours , at least 3 hours , at least 4 hours , at least 5 hours , at moiety can be linked via its N -terminus or C - terminus to the least 6 hours , at least 7 hours , at least 8 hours , at least 12 first portion of the intein sequence. The cleavage moiety can hours , at least 16 hours , at least 20 hours , at least 24 hours , comprise the second portion of the intein sequence . The first at least 2 days, at least 3 days, at least 4 days, at least 5 days, portion or second portion of the intein sequence can be the at least 6 days , at least 1 week , at least 2 weeks , at least 3 40 N - terminal intein , the C - terminal intein , or any other suit weeks, at least 1 month or longer . able portion of an intein that can facilitate release of the A chimeric receptor polypeptide can bind any suitable actuator moiety . The intein sequences can be from any antigen as described herein . In some embodiments , the suitable source . The first and second portion can be from the chimeric receptor polypeptide is a transmembrane receptor. same or different sources ( e . g . , organism , protein ). In an In some embodiments , the chimeric receptor polypeptide is 45 illustrative example , an actuator moiety can be covalently an intracellular receptor . In some embodiments , the chimeric linked ( e . g . , at its N - terminus or C - terminus ) via a peptide receptor polypeptide is a nuclear receptor. The antigen bond to a first portion of the intein sequence , which com interacting domain of a chimeric receptor polypeptide can prises an N -terminal intein . The actuator moiety - N -terminal bind a membrane bound antigen , for example an antigen intein fusion can be contacted with a second portion of the bound to the extracellular surface of a cell ( e . g . , a target 50 intein sequence comprising a C - terminal intein . This con cell ) . In some embodiments , the antigen interacting domain tacting of the first and second portion of the intein sequences binds a non -membrane bound antigen , for example an can result in a site specific cleavage ( e . g ., at a site between extracellular antigen that is secreted by a cell ( e . g . , a target the actuator moiety and the N - terminal intein ) , thereby cell) or an antigen located in the cytoplasm of a cell ( e. g ., a releasing the actuator moiety . In another illustrative target cell ) . Antigens (e . g ., membrane bound and non - 55 example , an actuator moiety can be covalently linked ( e . g . , membrane bound ) can be associated with a disease such as at its N -terminus or C - terminus ) via a peptide bond to a first a viral, bacterial, and /or parasitic infection ; inflammatory portion of the intein comprising a C - terminal intein . The and / or autoimmune disease ; or neoplasm such as a cancer actuator moiety - C -terminal intein fusion can be contacted and / or tumor. with a second portion of the intein sequence comprising an Upon exposure to the antigen , the chimeric receptor can 60 N - terminal intein . This contacting of the first and second undergo receptor modification . Receptor modification can portion of the inteins can result in a site - specific cleavage comprise a conformational change , chemical modification , (e . g ., at a suitable site between the actuator moiety and the or combination thereof. A chemical modification can com C -terminal intein ) , thereby releasing the actuator moiety . prise , for example , phosphorylation or dephosphorylation of In some embodiments, the cleavage recognition site com at least one amino acid residue of the receptor. Phosphory - 65 prises a disulfide bond . In some embodiments, the cleavage lation and / or dephosphorylation can occur at, for example , a moiety comprises oxidoreductase activity . The disulfide tyrosine , serine, threonine , or any other suitable amino acid bond can link the actuator moiety to a portion of the US 9 ,856 , 497 B2 79 80 chimeric adaptor polypeptide or chimeric receptor polypep - (Cas ) polypeptides, type IV CRISPR - associated (Cas ) poly tide . The disulfide bond can be formed by one or more peptides , type V CRISPR -associated (Cas ) polypeptides , cysteines of the actuator moiety . The cysteines can be and type VI CRISPR - associated (Cas ) polypeptides ; zinc engineered into the actuator moiety . The cysteines can be a finger nucleases ( ZFN ) ; transcription activator- like effector part of the native or wild - type sequence of the actuator 5 nucleases ( TALEN ) ; meganucleases ; RNA - binding proteins moiety . The cysteines can be present in a linker peptide (RBP ) ; CRISPR -associated RNA binding proteins ; recom appended to the actuator moiety . Cleavage of the disulfide binases ; flippases ; transposases ; Argonaute proteins ( e . g . , bond can be facilitated by, for example , altering the redox prokaryotic Argonaute (pAgo ) , archaeal Argonaute (aAgo ) , conditions of the disulfide bond . Alteration of the redox and eukaryotic Argonaute ( eAgo )) ; any derivative thereof conditions can lead to reduction of the disulfide bond to 10 any variant thereof and any fragment thereof. thiols and release of the actuator moiety . Cleavage of the In some embodiments , the actuator moiety comprises a disulfide bond can be facilitated by a cleavage moiety Cas protein that forms a complex with a guide nucleic acid , comprising a redox agent that can lead to reduction of the such as a guide RNA . In some embodiments, the actuator disulfide bond . The redox agent can be an enzyme, or any moiety comprises a RNA - binding protein (RBP ) optionally derivative , variant or fragment thereof. The enzyme can be 15 complexed with a guide nucleic acid , such as a guide RNA , an oxidoreductase . Examples of oxidoreductases include which is able to form a complex with a Cas protein . In some protein -disulfide reductase , thioredoxins, glutaredoxins, embodiments , the actuator moiety comprises a Cas protein thiol disulfide oxidoreductases ( e . g . , DsbA , BdbA - D , lacking cleavage activity . MdbA , SdbA ) , and glutathione disulfide reductase . The ThThe actuator moiety of a subject system can bind to a redox agent can be from any suitable source including 20 target polynucleotide to regulate expression and / or activity prokaryotes and eukaryotes. Cofactors ( e . g , nicotinamide of the target polynucleotide by physical obstruction of the cofactors , flavins , and derivatives and analogs thereof ) can target polynucleotide or recruitment of additional factors be supplied for optimal activity of the enzyme . effective to suppress or enhance expression of the target A GMP, as described elsewhere herein , can comprise an polynucleotide . In some embodiments , the actuator moiety actuator moiety linked to a cleavage recognition site . The 25 comprises a transcriptional activator effective to increase actuator moiety can comprise a nuclease ( e . g. , DNA nucle - expression of the target polynucleotide . The actuator moiety ase and / or RNA nuclease ), modified nuclease ( e . g ., DNA can comprise a transcriptional repressor effective to decrease nuclease and / or RNA nuclease ) that is nuclease - deficient or expression of the target polynucleotide . has reduced nuclease activity compared to a wild - type In some embodiments , the target polynucleotide com nuclease , a variant thereof, a derivative thereof, or a frag - 30 prises genomic DNA . In some embodiments , the target ment thereof as described elsewhere herein . The actuator polynucleotide comprises a region of a plasmid , for example moiety can regulate expression and /or activity of a gene or a plasmid carrying an exogenous gene . In some embodi edit the sequence of a nucleic acid (e . g ., a gene and /or gene ments , the target polynucleotide comprises RNA , for product) . In some embodiments , the actuator moiety com example mRNA . In some embodiments , the target poly prises a DNA nuclease such as an engineered ( e . g ., pro - 35 nucleotide comprises an endogenous gene or gene product. grammable or targetable ) DNA nuclease to induce genome The actuator moiety can include one or more copies of a editing of a target DNA sequence . In some embodiments , the nuclear localization signal that allows the actuator to trans actuator moiety comprises a RNA nuclease such as an locate into the nucleus upon cleavage from the GMP. engineered ( e . g ., programmable or targetable ) RNA nucle - A target polynucleotide of the various embodiments of the ase to induce editing of a target RNA sequence . In some 40 aspects herein can be DNA or RNA ( e . g . , mRNA ) . The embodiments , the actuator moiety has reduced or minimal target polynucleotide can be single -stranded or double nuclease activity . An actuator moiety having reduced or stranded . The target polynucleotide can be genomic DNA . minimal nuclease activity can regulate expression and / or The target polynucleotide can be any polynucleotide endog activity by physical obstruction of a target polynucleotide or enous or exogenous to a cell . For example , the target recruitment of additional factors effective to suppress or 45 polynucleotide can by a polynucleotide residing in the enhance expression of the target polynucleotide . In some nucleus of a eukaryotic cell . The target polynucleotide can embodiments , the actuator moiety comprises a nuclease -null be a sequence coding a gene product ( e . g ., a protein ) or a DNA binding protein derived from a DNA nuclease that can non - coding sequence ( e . g . , a regulatory polynucleotide ) . In induce transcriptional activation or repression of a target some embodiments, the target polynucleotide comprises a DNA sequence . In some embodiments , the actuator moiety 50 region of a plasmid , for example a plasmid carrying an comprises a nuclease -null RNA binding protein derived exogenous gene . In some embodiments , the target poly from a RNA nuclease that can induce transcriptional acti - nucleotide comprises RNA , for example mRNA . In some vation or repression of a target RNA sequence . In some embodiments , the target polynucleotide comprises an embodiments , the actuator moiety is a nucleic acid - guided endogenous gene or gene product . actuator moiety . In some embodiments, the actuator moiety 55 The target polynucleotide may include a number of dis is a DNA - guided actuator moiety . In some embodiments, the ease -associated genes and polynucleotides as well as sig actuator moiety is an RNA - guided actuator moiety . An naling biochemical pathway -associated genes and poly actuator moiety can regulate expression or activity of a gene nucleotides. Examples of target polynucleotides include a and / or edit a nucleic acid sequence , whether exogenous or sequence associated with a signaling biochemical pathway, endogenous . Upon cleavage of the cleavage recognition site , 60 e . g . , a signaling biochemical pathway - associated gene or the actuator moiety is activated to complex with a target polynucleotide . Examples of target polynucleotides include polynucleotide . a disease associated gene or polynucleotide . A " disease Any suitable nuclease can be used in an actuator moiety . associated ” gene or polynucleotide refers to any gene or Suitable nucleases include, but are not limited to , CRISPR polynucleotide which is yielding transcription or translation associated ( Cas) proteins or Cas nucleases including type I 65 products at an abnormal level or in an abnormal form in cells CRISPR - associated ( Cas ) polypeptides , type II CRISPR - derived from a disease -affected tissue compared with tissue associated ( Cas ) polypeptides, type III CRISPR -associated (s ) or cells of a non -disease control. In some embodiments , US 9 , 856 , 497 B2 81 82 it is a gene that becomes expressed at an abnormally high Genetic Medicine, Johns Hopkins University (Baltimore , level. In some embodiments , it is a gene that becomes Md. ) and National Center for Biotechnology Information , expressed at an abnormally low level . The altered expression National Library of Medicine (Bethesda , Md. ) , available on can correlate with the occurrence and /or progression of the the World Wide Web . Exemplary genes associated with disease. A disease -associated gene also refers to a gene 5 possessing mutation ( s ) or genetic variation that is directlygene 5 certain diseases and disorders are provided in Tables 3 and responsible or is in linkage disequilibrium with a gene ( s ) 4 . Examples of signaling biochemical pathway - associated that is response for the etiology of a disease. The transcribed genes and polynucleotides are listed in Table 5 . or translated products may be known or unknown , and may be at a normal or abnormal level. Mutations in these genes and pathways can result in Examples of disease - associated genes and polynucle - production of improper proteins or proteins in improper otides are available from McKusick -Nathans Institute of amounts which affect function . TABLE 3 DISEASE /DISORDERS GENE( S ) Neoplasia PTEN ; ATM ; ATR ; EGFR ; ERBB2; ERBB3 ; ERBB4 ; Notch1; Notch2 ; Notch3; Notch4 ; AKT; AKT2; AKT3 ; HIF ; HIF1a ; HIF3a ; Met ; HRG ; Bc12 ; PPAR alpha ; PPAR gamma; WT1 (Wilms Tumor ) ; FGF Receptor Family members ( 5 members : 1 , 2 , 3 , 4 , 5 ) ; CDKN2a ; APC ; RB (retinoblastoma ) ; MEN1; VHL ; BRCA1; BRCA2; AR ( Androgen Receptor ) ; TSG101 ; IGF ; IGF Receptor; Igfl ( 4 variants ) ; Igf2 ( 3 variants ) ; Igf 1 Receptor; Igf 2 Receptor; Bax ; Bc12 ; caspases family ( 9 members : 1 , 2 , 3 , 4 , 6 , 7 , 8 , 9 , 12 ) ; Kras ; Apc Age - related Macular Abcr; Ccl2 ; Cc2 ; cp (ceruloplasmin ); Timp3 ; cathepsinD ; Degeneration Vldlr ; Cer2 Neuregulin1 (Nrgl ) ; Erb4 ( receptor for Neuregulin ) ; Complexinl (Cplxl ) ; Tph1 hydroxylase ; Tph2 Tryptophan hydroxylase 2 ; Neurexin 1 ; GSK3 ; GSK3a; GSK3b Disorders 5 - HTT ( Slc6a4 ) ; COMT ; DRD ( Drdla ); SLC6A3; DAOA ; DTNBP1; Dao (Daol ) Trinucleotide Repeat HTT ( Huntington ' s Dx ) ; SBMA/ SMAX1/ AR (Kennedy ' s Disorders Dx ) ; FXN /X25 ( Friedrich ' s Ataxia ) ; ATX3 (Machado Joseph ' s Dx ) ; ATXN1 and ATXN2 (spinocerebellar ataxias ); DMPK ( ) ; Atrophin - 1 and Atn1 (DRPLA Dx ); CBP ( Creb - BP - global instability ) ; VLDLR ( Alzheimer ' s ) ; Atxn7 ; Atxn10 Fragile X Syndrome FMR2; FXR1; FXR2 ; mGLUR5 Secretase Related Disorders APH - 1 ( alpha and beta ) ; Presenilin (Psenl ); nicastrin (Nestn ) ; PEN - 2 Others Nosl ; Parp1 ; Nat1 ; Nat2 Prion - related disorders Prp ALS SOD1; ALS2; STEX ; FUS ; TARDBP ; VEGF ( VEGF -a ; VEGF - b ; VEGF - C ) Drug Prkce (alcohol ) ; Drd2 ; Drd4 ; ABAT (alcohol ) ; GRIA2; Grm5 ; Grinl ; Htrlb ; Grin2a ; Drd3 ; Pdyn ; Grial (alcohol ) Autism Mecp2 ; BZRAP1; MDGA2; Sema5A ; Neurexin 1 ; Fragile X ( FMR2 (AFF2 ) ; FXR1 ; FXR2 ; Mglur5 ) Alzheimer ' s Disease E1; CHIP ; UCH ; UBB ; Tau ; LRP ; PICALM ; Clusterin ; PS1 ; SORL1 ; CR1; Vldlr ; Ubal ; Uba3; CHIP28 (Aqpl , Aquaporin 1 ) ; Uchli ; Uch13 ; APP Inflammation IL - 10 ; IL - 1 ( IL - 1a ; IL - 1b ) ; IL - 13; IL - 17 ( IL - 17a ( CTLA8 ) ; IL 17b ; IL - 17c; IL - 17d ; IL -17f ) ; II -23 ; Cx3cri ; ptpn22 ; TNFa; NOD2/ CARD15 for IBD ; IL - 6 ; IL - 12 ( IL - 12a ; IL - 12b ) ; CTLA4 ; Cx3cli Parkinson 's Disease x -Synuclein ; DJ- 1 ; LRRK2; Parkin ; PINK1

TABLE 4 Blood and Anemia (CDAN1 , CDA1, RPS19 , DBA , PKLR , PK1, NT5C3 , UMPH1, coagulation PSN1 , RHAG , RH50A , NRAMP2 , SPTB , ALAS2 , ANH1, ASB , diseases and ABCB7, ABC7, ASAT ) ; Bare lymphocyte syndrome ( TAPBP , TPSN , disorders TAP2 , ABCB3 , PSF2 , RING11, MHC2TA , C2TA , RFX5 , RFXAP, RFX5) , Bleeding disorders ( TBXA2R , P2RX1, P2X1) ; Factor H and factor H - like 1 (HF1 , CFH , HUS) ; Factor V and factor VIII (MCFD2 ) ; Factor VII deficiency ( F7) ; Factor X deficiency (F10 ) ; Factor XI deficiency ( F11 ) ; Factor XII deficiency ( F12 , HAF ) ; Factor XIIIA deficiency (F13A1 , F13A ) ; Factor XIIIB deficiency ( F13B ) ; Fanconi anemia (FANCA , FACA , FA1, FA , FAA , FAAP95 , FAAP90 , FLJ34064 , FANCB , FANCC , FACC , BRCA2, FANCD1, FANCD2, FANCD , FACD , FAD , FANCE , FACE , FANCF , XRCC9 , FANCG , BRIP1, BACH1, FANCJ, PHF9 , FANCL , FANCM , KIAA1596 ) ; Hemophagocytic lymphohistiocytosis disorders (PRF1 , HPLH2 , US 9 , 856 , 497 B2 83 84 TABLE 4 -continued UNC13D , MUNC13 - 4 , HPLH3, HLH3, FHL3 ) ; Hemophilia A ( F8 , F8C , HEMA ) ; Hemophilia B ( F9 , HEMB ) , Hemorrhagic disorders ( PI , ATT , F5 ) ; Leukocyde deficiencies and disorders (ITGB2 , CD18 , LCAMB , LAD , EIF2B1, EIF2BA , EIF2B2 , EIF2B3 , EIF2B5 , LVWM , CACH , CLE , EIF2B4 ) ; Sickle cell anemia (HBB ) ; Thalassemia (HBA2 , HBB , HBD , LCRB , HBA1) . Cell dysregulation B - cell non -Hodgkin lymphoma ( BCL7A , BCL7 ) ; Leukemia ( TAL1, and oncology TCL5 , SCL , TAL2 , FLT3 , NBS1, NBS , ZNFN1A1, IK1, LYF1, diseases and HOXD4, HOX4B , BCR , CML , PHL , ALL , ARNT, KRAS2 , RASK2, disorders GMPS, AF10 , ARHGEF12 , LARG , KIAA0382 , CALM , CLTH , CEBPA , CEBP, CHIC2 , BTL , FLT3 , KIT , PBT, LPP, NPM1, NUP214 , D9S46E , CAN , CAIN , RUNX1, CBFA2 , AML1, WHSC1L1, NSD3, FLT3 , AF1Q , NPM1, NUMAI, ZNF145 , PLZF, PML , MYL , STATSB , AF10 , CALM , CLTH , ARL11, ARLTS1 , P2RX7, P2X7, BCR , CML, PHL , ALL , GRAF , NF1, VRNF, WSS , NFNS, PTPN11 , PTP2C , SHP2, NS1 , BCL2 , CCND1, PRAD1, BCL1, TCRA , GATAI, GF1 , ERYF1, NFE1, ABL1 , NQO1, DIA4 , NMORI, NUP214 , D9S46E , CAN , CAIN ) . Inflammation and AIDS (KIR3DL1 , NKAT3 , NKB1 , AMB11 , KIR3DS1 , IFNG , CXCL12 , immune related SDF1) ; Autoimmune lymphoproliferative syndrome ( TNFRSF6 , APT1 , diseases and FAS , CD95 , ALPS1A ) ; Combined immunodeficiency , ( IL2RG , disorders SCIDX1, SCIDX , IMD4) ; HIV - 1 (CCL5 , SCYA5 , D175136E , TCP228 ) , HIV susceptibility or infection ( IL10 , CSIF , CMKBR2 , CCR2 , CMKBR5 , CCCKR5 ( CCR5 ) ) ; Immunodeficiencies (CD3E , CD3G , AICDA , AID , HIGM2, TNFRSF5 , CD40 , UNG , DGU , HIGM4, TNFSF5 , CD40LG , HIGM1, IGM , FOXP3 , IPEX , AIID , XPID , PIDX , TNFRSF14B , TACI) ; Inflammation ( IL - 10 , IL - 1 ( IL -1a , IL - 1b ) , IL - 13 , IL - 17 (IL - 17a (CTLAS ) , IL - 17b, IL - 17c, IL - 17d , IL - 17f) , II - 23 , Cx3cr1, ptpn22 , TNFa , NOD2 /CARD15 for IBD , IL - 6 , IL - 12 ( IL - 12a , IL - 12b ) , CTLA4, Cx3cli ) ; Severe combined immunodeficiencies ( SCIDs) ( JAK3 , JAKL , DCLREIC , ARTEMIS , SCIDA , RAG1, RAG2, ADA , PTPRC , CD45 , LCA , IL7R , CD3D , T3D , IL2RG , SCIDX1, SCIDX , IMD4 ) . Metabolic , liver , Amyloid neuropathy ( TTR , PALB ) ; Amyloidosis ( APOA1, APP , AAA , kidney and CVAP , AD1, GSN , FGA , LYZ , TTR , PALB ); Cirrhosis (KRT18 , KRT8 , protein diseases CIRH1A , NAIC , TEX292 , KIAA1988 ); Cystic fibrosis (CFTR , ABCC7, and disorders CF, MRP7 ); Glycogen storage diseases (SLC2A2 , GLUT2 , G6PC , G6PT, G6PT1, GAA , LAMP2 , LAMPB , AGL , GDE , GBE1 , GYS2 , PYGL , PFKM ) ; Hepatic adenoma, 142330 ( TCF1, HNF1A , MODY3) , Hepatic failure , early onset, and neurologic disorder ( SCOD1, SCO1) , Hepatic lipase deficiency ( LIPC ) , Hepatoblastoma, cancer and (CTNNB1 , PDGFRL , PDGRL , PRLTS , AXIN1 , AXIN , CTNNB1, TP53 , P53 , LFS1 , IGF2R , MPRI, MET, CASP8 , MCH5 ; Medullary cystic kidney disease (UMOD , HNFJ, FJHN , MCKD2 , ADMCKD2) ; Phenylketonuria (PAH , PKU1, QDPR , DHPR , PTS ) ; Polycystic kidney and hepatic disease (FCYT , PKHD1, ARPKD , PKD1, PKD2, PKD4 , PKDTS , PRKCSH , G19P1 , PCLD , SEC63 ) . Muscular/ Skeletal Becker muscular dystrophy ( DMD , BMD , MYF6 ) , Duchenne Muscular diseases and Dystrophy (DMD , BMD ); Emery -Dreifuss muscular dystrophy (LMNA , disorders LMN1, EMD2, FPLD , CMD1A , HGPS , LGMD1B , LMNA , LMN1 , EMD2 , FPLD , CMD1A ); Facioscapulohumeral muscular dystrophy ( FSHMD1A , FSHD1A ) ; Muscular dystrophy (FKRP , MDCIC , LGMD2I , LAMA2 , LAMM , LARGE , KIAA0609 , MDC1D , FCMD , TTID , MYOT, CAPN3 , CANP3 , DYSF , LGMD2B , SGCG , LGMD2C , DMDA1, SCG3, SGCA , ADL , DAG2 , LGMD2D , DMDA2, SGCB , LGMD2E , SGCD , SGD , LGMD2F , CMD1L , TCAP, LGMD2G , CMD1N , TRIM32 , HT2A , LGMD2H , FKRP, MDC1C , LGMD21, TTN , CMD1G , TMD , LGMD2J, POMT1 , CAV3, LGMD1C , SEPN1, SELN , RSMD1, PLEC1, PLTN , EBS1 ) ; Osteopetrosis (LRP5 , BMND1, LRP7 , LR3 , OPPG , VBCH2, CLCN7, CLC7 , OPTA2, OSTMI, GL , TCIRG1, TIRC7 , OC116 , OPTB1) ; Muscular atrophy (VAPB , VAPC , ALS8 , SMN1 , SMA1, SMA2 , SMA3 , SMA4 , BSCL2 , SPG17 , GARS , SMADI, CMT2D , HEXB , IGHMBP2, SMUBP2 , CATF1, SMARD1) . Neurological and ALS (SOD1 , ALS2 , STEX , FUS , TARDBP , VEGF (VEGF - a , VEGF - b , neuronal diseases VEGF - c ) ; Alzheimer disease (APP , AAA , CVAP, AD1, APOE , AD2, and disorders PSEN2 , AD4 , STM2, APBB2, FE65L1, NOS3, PLAU , URK , ACE , DCP1, ACE1 , MPO , PACIP1 , PAXIPIL , PTIP , A2M , BLMH , BMH , PSEN1, AD3 ) ; Autism (Mecp2 , BZRAP1 , MDGA2, Sema5A , Neurexinl , GLO1, MECP2, RTT, PPMX , MRX16 , MRX79 , NLGN3, NLGN4 , KIAA1260 , AUTSX2) ; Fragile X Syndrome (FMR2 , FXR1 , FXR2, mGLUR5 ) ; Huntington ' s disease and disease like disorders (HD , IT15 , PRNP, PRIP , JPH3, JP3 , HDL2, TBP, SCA17 ) ; Parkinson disease (NR4A2 . NURR1. NOT, TINUR , SNCAIP , TBP , SCA17 , SNCA , NACP, PARK1, PARK4 , DJ1, PARK7 , LRRK2 , PARKS, PINK1 , PARK6, UCHL1 , PARK5 , SNCA , NACP, PARK1 , PARK4 , PRKN , PARK2, PDJ, DBH , NDUFV2) ; Rett syndrome (MECP2 , RTT, PPMX , MRX16 , MRX79 , CDKL5 , STK9 , MECP2 , RTT, PPMX , MRX16 , MRX79 , x - Synuclein , DJ - 1 ) ; Schizophrenia (Neuregulin1 (Nrgl ) , Erb4 ( receptor for Neuregulin ) , Complexinl ( Cplx1) , Tph1 Tryptophan hydroxylase , Tph2 , Tryptophan hydroxylase 2 , Neurexin 1 , GSK3 , GSK3a, GSK3b , 5 - HTT ( Slc6a4 ) , COMT, DRD ( Drdla) , SLC6A3 , DAOA , DTNBP1, Dao (Daol ) ) ; Secretase Related Disorders ( APH - 1 US 9 , 856 , 497 B2 85 86 TABLE 4 -continued (alpha and beta ) , Presenilin (Psen1 ) , nicastrin , (Nestn ) , PEN - 2 , Nos1, Parpl , Nati , Nat2 ) ; Trinucleotide Repeat Disorders (HTT (Huntington ' s Dx ) , SBMA / SMAX1/ AR (Kennedy ' s Dx ) , FXN /X25 ( Friedrich ' s Ataxia ), ATX3 (Machado - Joseph ' s Dx ) , ATXN1 and ATXN2 (spinocerebellar ataxias ) , DMPK (myotonic dystrophy ) , Atrophin - 1 and Atn1 ( DRPLA Dx ), CBP ( Creb - BP - global instability ) , VLDLR ( Alzheimer 's ) , Atxn7 , Atxn10 ) . Occular diseases Age - related macular degeneration ( Abcr , Ccl2 , Cc2 , cp (ceruloplasmin ) , and disorders Timp3 , cathepsinD , Vldlr, Ccr2 ) ; Cataract (CRYAA , CRYA1 , CRYBB2 , CRYB2, PITX3, BFSP2 , CP49 , CP47 , CRYAA , CRYAI , PAX6 , AN2 , MGDA , CRYBAI, CRYB1, CRYGC , CRYG3, CCL , LIM2, MP19 , CRYGD , CRYG4, BFSP2 , CP49 , CP47 , HSF4 , CTM , HSF4 , CTM , MIP , AQPO , CRYAB , CRYA2, CTPP2, CRYBB1, CRYGD , CRYG4, CRYBB2, CRYB2 , CRYGC , CRYG3 , CCL , CRYAA , CRYA1, GJAS, CX50, CAE1 , GJA3 , CX46 , CZP3 , CAE3 , CCM1, CAM , KRIT1 ) ; Corneal clouding and dystrophy ( APOA1, TGFBI, CSD2, CDGG1, CSD , BIGH3 , CDG2, TACSTD2, TROP2 , Misi, VSX1, RINX , PPCD , PPD , KTCN , COL8A2 , FECD , PPCD2, PIP5K3, CFD ) ; Cornea plana congenital ( KERA , CNA2 ) ; (MYOC , TIGR , GLC1A , JOAG , GPOA , OPTN , GLC1E , FIP2, HYPL , NRP, CYP1B1, GLC3A , OPA1, NTG , NPG , CYP1B1 , GLC3A ) ; Leber congenital amaurosis ( CRB1 , RP12 , CRX , CORD2 , CRD , RPGRIP1 , LCA6 , CORDO, RPE65 , RP20 , AIPL1 , LCA4 , GUCY2D , GUC2D , LCAI , CORD6 , RDH12 , LCA3 ) ; Macular dystrophy ( ELOVL4 , ADMD , STGD2, STGD3, RDS , RP7, PRPH2, PRPH , AVMD , AOFMD , VMD2 ).

TABLE 5 CELLULAR FUNCTION GENES PI3K /AKT Signaling PRKCE ; ITGAM ; ITGA5; IRAK1; PRKAA2 ; EIF2AK2; PTEN ; EIF4E ; PRKCZ ; GRK6 ; MAPK1; TSC1; ; AKT2 ; IKBKB ; PIK3CA ; CDK8 ; CDKN1B ; NFKB2; BCL2; PIK3CB ; PPP2R1A ; MAPK8; BCL2L1; MAPK3 ; TSC2; ITGA1; KRAS ; EIF4EBP1; RELA ; PRKCD ; NOS3 ; PRKAA1 ; MAPK9; CDK2; PPP2CA ; PIM1; ITGB7; YWHAZ ; ILK ; TP53 ; RAF1 ; IKBKG ; RELB ; DYRK1A ; CDKN1A ; ITGB1 ; MAP2K2; JAK1; AKT1 ; JAK2; PIK3R1; CHUK ; PDPK1; PPP2R5C ; CTNNB1; MAP2K1; NFKB1; PAK3 ; ITGB3; CCND1; GSK3A ; FRAP1 ; SFN ; ITGA2 ; TTK ; CSNK1A1 ; BRAF ; GSK3B ; AKT3; FOXO1; SGK ; HSP90AA1; RPS6KB1 ERK /MAPK Signaling PRKCE ; ITGAM ; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2 ; RAC1; RAP1A ; TLN1; EIF4E ; ELK1; GRK6 ; MAPK1: RAC2: PLK1: AKT2 : PIK3CA : CDK8 : CREB1 : PRKCI; PTK2; FOS ; RPS6KA4 ; PIK3CB ; PPP2R1A ; PIK3C3 ; MAPK8; MAPK3; ITGA1 ; ETS1 ; KRAS ; MYCN ; EIF4EBP1 ; PPARG ; PRKCD ; PRKAA1; MAPK9 ; SRC ; CDK2; PPP2CA ; PIM1; PIK3C2A ; ITGB7; YWHAZ ; PPP1CC ; KSR1; PXN ; RAF1 ; FYN ; DYRK1A ; ITGB1; MAP2K2 ; PAK4 ; PIK3R1; STAT3 ; PPP2R5C ; MAP2K1 ; PAK3 ; ITGB3 ; ESR1; ITGA2; MYC ; TTK ; CSNK1A1; CRKL; BRAF ; ATF4; PRKCA ; SRF ; STAT1 ; SGK RAC1; TAF4B ; EP300 ; SMAD2; TRAF6 ; PCAF; ELK1; Signaling MAPK1; SMAD3 ; AKT2; IKBKB ; NCOR2 ; UBE21; PIK3CA ; CREB1 ; FOS; HSPA5 ; NFKB2 ; BCL2 ; MAP3K14 ; STAT5B ; PIK3CB ; PIK3C3; MAPK8; BCL2L1; MAPK3; TSC22D3 ; MAPK10 ; NRIP1 ; KRAS; MAPK13 ; RELA ; STAT5A ; MAPK9; NOS2A ; PBX1; NR3C1 ; PIK3C2A ; CDKN1C ; TRAF2 ; SERPINE1; NCOA3; MAPK14 ; TNF ; RAF1 ; IKBKG ; MAP3K7 ; CREBBP ; CDKN1A ; MAP2K2; JAK1; IL8 ; NCOA2; AKT1; JAK2; PIK3R1; CHUK ; STAT3 ; MAP2K1; NFKB1; TGFBR1; ESR1; SMAD4 ; CEBPB ; JUN ; AR ; AKT3 ; CCL2 ; MMP1; STAT1 ; IL6 ; HSP90AA1 Axonal Guidance Signaling PRKCE ; ITGAM ; ROCK1; ITGA5; CXCR4 ; ADAM12 ; IGF1; RAC1; RAP1A ; EIF4E ; PRKCZ ; NRP1; NTRK2; ARHGEF7 ; SMO ; ROCK2 ; MAPK1; PGF ; RAC2; PTPN11 ; GNAS; AKT2 ; PIK3CA ; ERBB2 ; PRKCI; PTK2; CFL1; GNAQ ; PIK3CB ; CXCL12 ; PIK3C3; WNT11 ; PRKD1 ; GNB2L1; ABL1; MAPK3; ITGA1; KRAS ; RHOA ; PRKCD ; PIK3C2A ; ITGB7 ; GLI2 ; PXN ; VASP ; RAF1 ; FYN ; ITGB1 ; MAP2K2 ; PAK4 ; ADAM17 ; AKT1 ; PIK3R1; GLI1 ; WNT5A ; ADAM10 ; MAP2K1; PAK3 ; ITGB3; CDC42 ; VEGFA ; ITGA2 ; EPHA8 ; CRKL ; ; GSK3B ; AKT3 ; PRKCA US 9 , 856 , 497 B2 87 88 TABLE 5 -continued CELLULAR FUNCTION GENES Signaling PRKCE ; ITGAM ; ROCK1; ITGA5 ; CXCR4 ; IRAK1 ; PRKAA2; EIF2AK2; RAC1; RAP1A ; GRK6 ; ROCK2; MAPK1; PGF ; RAC2 ; PTPN11 ; GNAS ; PLK1; AKT2 ; DOK1; CDK8 ; CREB1; PTK2; CFL1; GNAQ ; MAP3K14 ; CXCL12 ; MAPK8 ; GNB2L1 ; ABL1; MAPK3; ITGA1 ; KRAS; RHOA ; PRKCD ; PRKAA1; MAPK9 ; SRC ; CDK2; PIM1; ITGB7; PXN ; RAF1 ; FYN ; DYRK1A ; ITGB1; MAP2K2; PAK4 ; AKT1 ; JAK2; STAT3; ADAM10 ; MAP2K1; PAK3 ; ITGB3; CDC42; VEGFA ; ITGA2; EPHA8 ; TTK ; CSNK1A1; CRKL ; BRAF ; PTPN13 ; ATF4 ; AKT3; SGK Actin Cytoskeleton Signaling ACTN4; PRKCE ; ITGAM ; ROCK1; ITGA5 ; IRAK1; PRKAA2; EIF2AK2; RAC1; INS ; ARHGEF7 ; GRK6 ; ROCK2 ; MAPK1; RAC2; PLK1; AKT2 ; PIK3CA ; CDK8 ; PTK2 ; CFL1; PIK3CB ; MYH9 ; DIAPH1; PIK3C3; MAPK8 ; F2R ; MAPK3; SLC9A1; ITGA1; KRAS ; RHOA ; PRKCD ; PRKAA1 ; MAPK9; CDK2; PIM1; PIK3C2A ; ITGB7 ; PPP1CC ; PXN ; VIL2 ; RAF1 , GSN ; DYRK1A ; ITGB1 ; MAP2K2 ; PAK4 ; PIP5K1A ; PIK3R1; MAP2K1 ; PAK3 ; ITGB3 ; CDC42 ; APC ; ITGA2 ; TTK ; CSNK1A1 ; CRKL ; BRAF ; VAV3; SGK Huntington ' s Disease Signaling PRKCE ; IGF1 ; EP300 ; RCOR1; PRKCZ ; HDAC4 ; TGM2; MAPK1; CAPNS1; AKT2 ; EGFR ; NCOR2; SP1 ; CAPN2 ; PIK3CA ; HDAC5 ; CREB1 ; PRKCI; HSPA5 ; REST ; GNAQ ; PIK3CB ; PIK3C3; MAPK8 ; IGF1R ; PRKD1; GNB2L1 ; BCL2L1 ; CAPN1; MAPK3; CASP8 ; HDAC2; HDACTA ; PRKCD ; HDAC11 ; MAPK9 ; HDAC9 ; PIK3C2A ; HDAC3 ; TP53 ; CASP9 ; CREBBP ; AKT1 ; PIK3R1; PDPK1; CASP1; APAF1 ; FRAP1 ; CASP2 ; JUN ; BAX ; ATF4 ; AKT3 ; PRKCA ; CLTC; SGK ; HDAC6; CASP3 Apoptosis Signaling PRKCE ; ROCK1; BID ; IRAK1; PRKAA2 ; EIF2AK2; BAK1; BIRC4 ; GRK6 ; MAPK1 ; CAPNS1; PLK1; AKT2 ; IKBKB ; CAPN2 ; CDK8; FAS ; NFKB2; BCL2 ; MAP3K14 ; MAPK8 ; BCL2L1 ; CAPN1; MAPK3; CASP8; KRAS; RELA ; PRKCD ; PRKAA1; MAPK9; CDK2 ; PIM1; TP53 ; TNF ; RAF1 ; IKBKG ; RELB ; CASP9 ; DYRK1A ; MAP2K2 ; CHUK ; APAF1; MAP2K1; NFKB1; PAK3; LMNA ; CASP2 ; BIRC2; TTK ; CSNK1A1; BRAF ; BAX ; PRKCA ; SGK ; CASP3 ; BIRC3; PARP1 B Cell Receptor Signaling RAC1, PTEN ; LYN ; ELK1 ; MAPK1; RAC2; PTPN11 ; AKT2 ; IKBKB ; PIK3CA ; CREB1; SYK ; NFKB2; CAMK2A ; MAP3K14 ; PIK3CB ; PIK3C3 ; MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13 ; RELA ; PTPN6 ; MAPK9 ; EGR1; PIK3C2A ; BTK ; MAPK14 ; RAF1 ; IKBKG ; RELB ; MAP3K7 ; MAP2K2 ; AKT1 ; PIK3R1; CHUK ; MAP2K1; NFKB1 ; CDC42 ; GSK3A ; FRAP1 ; BCL6 ; BCL10 ; JUN ; GSK3B : ATF4 : AKT3 : VAV3; RPS6KB1 Leukocyte Extravasation ACTN4; CD44 ; PRKCE ; ITGAM ; ROCK1; CXCR4; CYBA ; Signaling RAC1; RAP1A ; PRKCZ ; ROCK2 ; RAC2 ; PTPN11 ; MMP14 ; PIK3CA ; PRKCI; PTK2; PIK3CB; CXCL12 ; PIK3C3 ; MAPK8; PRKD1; ABL1; MAPK10 ; CYBB ; MAPK13 ; RHOA ; PRKCD ; MAPK9 ; SRC ; PIK3C2A ; BTK ; MAPK14 ; NOX1; PXN ; VIL2; VASP ; ITGB1; MAP2K2; CTNND1; PIK3R1 ; CTNNB1; CLDN1; CDC42 ; F11R ; ITK ; CRKL ; VAV3; CTTN ; PRKCA ; MMP1 ; MMP9 Integrin Signaling ACTN4 ; ITGAM ; ROCK1; ITGA5; RAC1 ; PTEN ; RAP1A ; TLN1; ARHGEF7 ; MAPK1; RAC2; CAPNS1 ; AKT2 ; CAPN2 ; PIK3CA ; PTK2 ; PIK3CB ; PIK3C3; MAPK8 ; CAV1; CAPN1; ABL1 ; MAPK3; ITGA1; KRAS ; RHOA ; SRC ; PIK3C2A ; ITGB7 ; PPP1CC ; ILK ; PXN ; VASP ; RAF1 ; FYN ; ITGB1; MAP2K2 ; PAK4 ; AKT1; PIK3R1 ; TNK2; MAP2K1; PAK3; ITGB3 ; CDC42 ; ; ITGA2; CRKL ; BRAF ; GSK3B ; AKT3 Acute Phase Response IRAK1; SOD2; MYD88 ; TRAF6 ; ELK1; MAPK1; PTPN11 ; Signaling AKT2 ; IKBKB ; PIK3CA ; FOS ; NFKB2; MAP3K14 ; PIK3CB ; MAPK8; RIPK1; MAPK3; ILÁST ; KRAS ; MAPK13 ; ILOR ; RELA ; SOCS1 ; MAPK9 ; FTL ; NR3C1; TRAF2 ; SERPINE1 ; MAPK14 ; TNF ; RAF1 ; PDK1; IKBKG ; RELB ; MAP3K7 ; MAP2K2; AKT1; JAK2; PIK3R1; CHUK ; STAT3 ; MAP2K1; NFKB1; FRAP1; CEBPB ; JUN ; AKT3 ; IL1R1; IL6 PTEN Signaling ITGAM ; ITGA5 ; RAC1 ; PTEN ; PRKCZ ; BCL2L11 ; MAPK1; RAC2; AKT2 ; EGFR ; IKBKB ; CBL ; PIK3CA ; CDKN1B ; PTK2 ; NFKB2; BCL2; PIK3CB ; BCL2L1; MAPK3; ITGA1; KRAS ; ITGB7; ILK ; PDGFRB ; INSR ; RAF1 ; IKBKG ; CASP9 ; CDKN1A ; ITGB1 ; MAP2K2; AKT1 ; PIK3R1; CHUK , PDGFRA ; PDPK1 ; MAP2K1; US 9 , 856 , 497 B2 89 90 TABLE 5 -continued CELLULAR FUNCTION GENES NFKB1 ; ITGB3 ; CDC42 ; CCND1; GSKJA ; ITGA2; GSK3B ; AKT3; FOXO1; CASP3 ; RPS6KB1 p53 Signaling PTEN ; EP300 ; BBC3; PCAF ; FASN ; BRCA1; GADD45A ; BIRC5 ; AKT2 ; PIK3CA ; CHEK1; TP53INP1 ; BCL2; PIK3CB ; PIK3C3 ; MAPK8; THBS1 ; ATR ; BCL2L1 ; ; PMAIP1 ; CHEK2 ; TNFRSF10B ; TP73 ; RB1; HDAC9 ; CDK2 ; PIK3C2A ; MAPK14 ; TP53; LRDD ; CDKN1A ; HIPK2; AKT1 ; PIK3R1; RRM2B ; APAF1; CTNNB1; SIRT1 ; CCND1; PRKDC ; ATM ; SFN ; CDKN2A ; JUN ; SNAI2 ; GSK3B ; BAX ; AKT3 Aryl Hydrocarbon Receptor HSPB1; EP300 ; FASN ; TGM2; RXRA ; MAPK1; NQO1; Signaling NCOR2 ; SP1; ARNT ; CDKN1B ; FOS; CHEK1; SMARCA4 ; NFKB2 ; MAPK8; ALDH1A1; ATR ; E2F1 ; MAPK3; NRIP1 ; CHEK2; RELA ; TP73 ; GSTP1 ; RB1; SRC ; CDK2; AHR ; NFE2L2 ; NCOA3; TP53 ; TNF ; CDKN1A ; NCOA2; APAF1 ; NFKB1; CCND1; ATM ; ESR1; CDKN2A ; MYC ; JUN ; ESR2; BAX ; IL6; CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE ; EP300 ; PRKCZ ; RXRA ; MAPK1; NQO1; Signaling NCOR2 ; PIK3CA ; ARNT; PRKCI; NFKB2; CAMK2A ; PIK3CB ; PPP2R1A ; PIK3C3 ; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1 ; KRAS; MAPK13 ; PRKCD ; GSTP1; MAPK9 ; NOS2A ; ABCB1; AHR ; PPP2CA ; FTL ; NFE2L2; PIK3C2A ; PPARGC1A ; MAPK14 ; TNF ; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C ; MAP2K1; NFKB1 ; KEAP1 ; PRKCA ; EIF2AK3; IL6 ; CYP1B1; HSP90AA1 SAPK / JNK Signaling PRKCE ; IRAK1; PRKAA2 ; EIF2AK2; RAC1; ELK1; GRK6 ; MAPK1; GADD45A ; RAC2; PLK1; AKT2 ; PIK3CA ; FADD ; CDK8; PIK3CB ; PIK3C3 ; MAPK8; RIPK1; GNB2L1 ; IRS1; MAPK3 ; MAPK10 ; DAXX ; KRAS ; PRKCD ; PRKAA1 ; MAPK9; CDK2 ; PIM1; PIK3C2A ; TRAF2 ; TP53 ; LCK ; MAP3K7; DYRK1A ; MAP2K2 ; PIK3R1; MAP2K1; PAK3; CDC42 ; JUN ; TTK , CSNK1A1; CRKL ; BRAF ; SGK PPAr/ RXR Signaling PRKAA2 ; EP300 ; INS ; SMAD2 ; TRAF6 ; PPARA ; FASN ; RXRA ; MAPK1; SMAD3 ; GNAS; IKBKB ; NCOR2 ; ABCA1; GNAQ ; NFKB2; MAP3K14 ; STAT5B ; MAPK8 ; IRS1 ; MAPK3 ; KRAS ; RELA ; PRKAAL; PPARGC1A ; NCOA3; MAPK14 ; INSR ; RAF1 ; IKBKG ; RELB ; MAP3K7; CREBBP ; MAP2K2; JAK2; CHUK ; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN ; IL1R1; PRKCA ; IL6 ; HSP90AA1; ADIPOQ NF- KB Signaling IRAK1; EIF2AK2 ; EP300 ; INS ; MYD88 ; PRKCZ ; TRAF6 ; TBK1; AKT2 ; EGFR ; IKBKB ; PIK3CA ; BTRC ; NFKB2 ; MAP3K14 ; PIK3CB ; PIK3C3 ; MAPK8; RIPK1; HDAC2; KRAS ; RELA ; PIK3C2A ; TRAF2 ; TLR4 ; PDGFRB ; TNF ; INSR ; LCK ; IKBKG ; RELB ; MAP3K7; CREBBP ; AKT1 ; PIK3R1 ; CHUK ; PDGFRA ; NFKB1; TLR2; BCL10 ; GSK3B ; AKT3; TNFAIP3 ; IL1R1 Neuregulin Signaling ERBB4; PRKCE ; ITGAM ; ITGA5 ; PTEN ; PRKCZ ; ELK1; MAPK1; PTPN11; AKT2 ; EGFR ; ERBB2 ; PRKCI; CDKN1B ; STAT5B ; PRKD1; MAPK3 ; ITGA1; KRAS ; PRKCD ; STAT5A ; SRC ; ITGB7; RAF1; ITGB1 ; MAP2K2 ; ADAM17 ; AKT1; PIK3R1 ; PDPK1 ; MAP2K1; ITGB3 ; EREG ; FRAP1 ; PSEN1; ITGA2 ; MYC ; NRG1; CRKL ; AKT3 ; PRKCA ; HSP90AA1; RPS6KB1 Wnt & Beta catenin Signaling CD44 ; EP300 ; LRP6 ; DVL3 ; CSNK1E ; GJA1; SMO ; AKT2 ; PIN1; CDH1; BTRC ; GNAQ ; MARK2; PPP2R1A ; WNT11 ; SRC ; DKK1; PPP2CA ; SOX6 ; SFRP2 ; ILK ; LEF1 ; SOX9; TP53 ; MAP3K7; CREBBP ; TCF7L2 ; AKT1 ; PPP2R5C ; WNT5A ; LRP5 ; CTNNB1 ; TGFBR1; CCND1; GSK3A ; DVL1; APC ; CDKN2A ; MYC ; CSNK1A1 ; GSK3B ; AKT3 ; Insulin Receptor PTEN ; INS; EIF4E ; PTPN1; PRKCZ ; MAPK1; TSC1; PTPN11 ; AKT2 ; CBL ; PIK3CA ; PRKCI; PIK3CB ; PIK3C3 ; MAPK8 ; IRS1; MAPK3 ; TSC2; KRAS ; EIF4EBP1 ; SLC2A4 ; PIK3C2A ; PPP1CC ; INSR ; RAF1; FYN ; MAP2K2; JAK1; AKT1 ; JAK2 ; PIK3R1 ; PDPK1; MAP2K1; GSK3A ; FRAP1; CRKL ; GSK3B ; AKT3 ; FOXO1 ; SGK ; RPS6KB1 IL - 6 Signaling HSPB1; TRAF6 ; MAPKAPK2; ELK1; MAPK1; PTPN11 ; IKBKB ; FOS ; NFKB2; MAP3K14 ; MAPK8; MAPK3; MAPK10 ; ILOST; KRAS ; MAPK13 ; ILOR ; RELA ; SOCS1 ; MAPK9; ABCB1; TRAF2 ; MAPK14 ; TNF; RAF1 ; IKBKG ; RELB ; MAP3K7; MAP2K2 ; IL8 ; JAK2 ; CHUK ; STAT3 ; MAP2K1; NFKB1; CEBPB ; JUN ; IL1R1 ; SRF; ILO US 9 , 856 , 497 B2 91 TABLE 5 -continued CELLULAR FUNCTION GENES Hepatic Cholestasis PRKCE ; IRAK1; INS ; MYD88; PRKCZ ; TRAF6 ; PPARA ; RXRA ; IKBKB ; PRKCI; NFKB2; MAP3K14 ; MAPK8; PRKD1 ; MAPK10 ; RELA ; PRKCD ; MAPKO ; ABCB1; TRAF2 ; TLR4; TNF ; INSR ; IKBKG ; RELB ; MAP3K7 ; IL8; CHUK ; NR1H2 ; TJP2 ; NFKB1; ESR1 ; SREBF1; FGFR4 ; JUN ; IL1R1; PRKCA ; IL6 IGF- 1 Signaling IGF1 ; PRKCZ ; ELK1; MAPK1; PTPN11 ; NEDD4; AKT2 ; PIK3CA ; PRKCI; PTK2; FOS ; PIK3CB ; PIK3C3 ; MAPK8 ; IGF1R ; IRS1; MAPK3; IGFBP7; KRAS ; PIK3C2A ; YWHAZ ; PXN ; RAF1; CASP9 ; MAP2K2; AKT1 ; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN ; JUN ; CYR61; AKT3 ; FOXO1; SRF ; CTGF; RPS6KB1 NRF2 -Mediated Oxidative PRKCE ; EP300 ; SOD2; PRKCZ ; MAPK1; SQSTM1; Response NQO1; PIK3CA ; PRKCI; FOS; PIK3CB ; PIK3C3; MAPK8 ; PRKD1 ; MAPK3; KRAS ; PRKCD ; GSTP1 ; MAPK9 ; FTL ; NFE2L2 ; PIK3C2A ; MAPK14 ; RAF1; MAP3K7; CREBBP ; MAP2K2; AKT1 ; PIK3R1; MAP2K1; PPIB ; JUN ; KEAP1 ; GSK3B ; ATF4 ; PRKCA ; EIF2AK3; HSP90AA1 Hepatic Fibrosis /Hepatic EDN1; IGF1 ; KDR ; FLT1 ; SMAD2; FGFR1 ; MET; PGF; Stellate Cell Activation SMAD3 ; EGFR ; FAS ; CSF1 ; NFKB2 ; BCL2 ; MYH9; IGF1R ; ILOR ; RELA ; TLR4; PDGFRB ; TNF ; RELB ; IL8 ; PDGFRA ; NFKB1; TGFBR1; SMAD4; VEGFA ; BAX ; IL1R1; CCL2; HGF ; MMP1 ; STAT1; IL6 ; CTGF; MMP9 PPAR Signaling EP300 ; INS ; TRAF6 ; PPARA ; RXRA ; MAPK1 ; IKBKB ; NCOR2; FOS ; NFKB2; MAP3K14 ; STAT5B ; MAPK3 ; NRIP1; KRAS; PPARG ; RELA ; STAT5A ; TRAF2 ; PPARGC1A ; PDGFRB ; TNF; INSR ; RAF1; IKBKG ; RELB ; MAP3K7; CREBBP ; MAP2K2; CHUK , PDGFRA ; MAP2K1; NFKB1; JUN ; IL1R1; HSP90AA1 Fc Epsilon RI Signaling PRKCE ; RAC1; PRKCZ ; LYN ; MAPK1; RAC2 ; PTPN11 ; AKT2 ; PIK3CA ; SYK ; PRKCI; PIK3CB ; PIK3C3; MAPK8 ; PRKD1; MAPK3; MAPK10 ; KRAS ; MAPK13; PRKCD ; MAPK9; PIK3C2A ; BTK ; MAPK14 ; TNF ; RAF1 ; FYN ; MAP2K2; AKT1; PIK3R1 ; PDPK1; MAP2K1; AKT3 ; VAV3; PRKCA G -Protein Coupled Receptor PRKCE ; RAP1A ; RGS16 ; MAPK1; GNAS; AKT2 ; IKBKB ; Signaling PIK3CA ; CREB1 ; GNAQ ; NFKB2 ; CAMK2A ; PIK3CB ; PIK3C3 ; MAPK3 ; KRAS ; RELA ; SRC ; PIK3C2A ; RAF1; IKBKG ; RELB ; FYN ; MAP2K2; AKT1 ; PIK3R1 ; CHUK ; PDPK1 ; STAT3 ; MAP2K1 ; NFKB1 ; BRAF; ATF4 ; AKT3 ; PRKCA Phosphate Metabolism PRKCE ; IRAK1 ; PRKAA2 ; EIF2AK2 ; PTEN ; GRK6 ; MAPK1; PLK1; AKT2 ; PIK3CA ; CDK8 ; PIK3CB ; PIK3C3 ; MAPK8; MAPK3 ; PRKCD ; PRKAA1; MAPK9 ; CDK2 ; PIM1; PIK3C2A ; DYRK1A ; MAP2K2 ; PIP5K1A ; PIK3R1; MAP2K1; PAK3 ; ATM ; TTK ; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1; ABL2; MAPK1; PIK3CA ; FOS ; PIK3CB ; PIK3C3 ; MAPK8; CAV1; ABL1; MAPK3 ; KRAS ; SRC ; PIK3C2A ; PDGFRB ; RAF1; MAP2K2; JAK1; JAK2; PIK3R1 ; PDGFRA ; STAT3 ; SPHK1; MAP2K1; MYC ; JUN ; CRKL ; PRKCA ; SRF ; STAT1; SPHK2 VEGF Signaling ACTN4 ; ROCK1; KDR ; FLT1 ; ROCK2; MAPK1; PGF ; AKT2 ; PIK3CA ; ARNT; PTK2 ; BCL2 ; PIK3CB ; PIK3C3 ; BCL2L1; MAPK3; KRAS ; HIF1A ; NOS3 ; PIK3C2A ; PXN ; RAF1 ; MAP2K2; ELAVL1; AKT1; PIK3R1 ; MAP2K1; SFN ; VEGFA ; AKT3; FOXO1; PRKCA Natural Killer Cell Signaling PRKCE ; RAC1; PRKCZ ; MAPK1; RAC2 ; PTPN11 ; KIR2DL3; AKT2 ; PIK3CA ; SYK ; PRKCI; PIK3CB ; PIK3C3 ; PRKD1; MAPK3 ; KRAS ; PRKCD ; PTPN6 ; PIK3C2A ; LCK ; RAF1 ; FYN ; MAP2K2; PAK4; AKT1 ; PIK3R1 ; MAP2K1; PAK3; AKT3 ; VAV3 ; PRKCA Cell Cycle : G1/ S Checkpoint HDAC4 ; SMAD3 ; SUV39H1; HDAC5; CDKN1B ; BTRC ; Regulation ATR : ABL1; E2F1; HDAC2; HDAC7A ; RB1 ; HDAC11; HDAC9 ; CDK2; ; HDAC3 ; TP53; CDKN1A ; CCND1; ; ATM ; RBL2 ; SMAD4 ; CDKN2A ; MYC ; NRG1; GSK3B ; RBL1; HDAC6 T Cell Receptor Signaling RAC1; ELK1; MAPK1; IKBKB ; CBL ; PIK3CA ; FOS ; NFKB2 ; PIK3CB ; PIK3C3 ; MAPK8; MAPK3; KRAS; RELA ; PIK3C2A ; BTK ; LCK ; RAF1 ; IKBKG ; RELB ; FYN ; MAP2K2; PIK3R1; CHUK ; MAP2K1; NFKB1; ITK ; BCL10 ; JUN ; VAV3 Death Receptor Signaling CRADD ; HSPB1; BID ; BIRC4 ; TBK1; IKBKB ; FADD ; FAS; NFKB2; BCL2 ; MAP3K14 ; MAPK8 ; RIPK1; CASP8 ; DAXX ; TNFRSF10B ; RELA ; TRAF2 ; TNF; IKBKG ; RELB ; CASP9 ; CHUK ; APAF1 ; NFKB1 ; CASP2 ; BIRC2; CASP3 ; BIRC3 FGF Signaling RAC1; FGFR1 ; MET; MAPKAPK2; MAPK1 ; PTPN11 ; AKT2 ; PIK3CA ; CREB1 ; PIK3CB ; PIK3C3; MAPK8; US 9 , 856 , 497 B2 93 94 TABLE 5 -continued CELLULAR FUNCTION GENES MAPK3; MAPK13 ; PTPN6 ; PIK3C2A ; MAPK14 ; RAF1 ; AKT1 ; PIK3R1; STAT3 ; MAP2K1; FGFR4 ; CRKL ; ATF4 ; AKT3 ; PRKCA ; HGF GM - CSF Signaling LYN ; ELK1; MAPK1; PTPN11 ; AKT2 ; PIK3CA ; CAMK2A ; STAT5B ; PIK3CB ; PIK3C3 ; GNB2L1 ; BCL2L1 ; MAPK3; ETS1 ; KRAS; RUNX1 ; PIM1; PIK3C2A ; RAF1; MAP2K2; AKT1 ; JAK2; PIK3R1; STAT3 ; MAP2K1; CCND1; AKT3 ; STAT1 Amyotrophic Lateral Sclerosis BID ; IGF1 ; RAC1; BIRC4 ; PGF ; CAPNS1 ; CAPN2; Signaling PIK3CA ; BCL2; PIK3CB ; PIK3C3 ; BCL2L1; CAPN1; PIK3C2A ; TP53 ; CASP9 ; PIK3R1; RAB5A ; CASP1; APAF1; VEGFA ; BIRC2; BAX ; AKT3 ; CASP3 ; BIRC3 JAK / Stat Signaling PTPN1; MAPK1 ; PTPN11; AKT2 ; PIK3CA ; STAT5B ; PIK3CB ; PIK3C3; MAPK3; KRAS ; SOCS1 ; STAT5A ; PTPN6 ; PIK3C2A ; RAF1 ; CDKN1A ; MAP2K2; JAK1; AKT1 ; JAK2; PIK3R1; STAT3 ; MAP2K1; FRAP1 ; AKT3 ; STAT1 Nicotinate and Nicotinamide PRKCE ; IRAK1 ; PRKAA2; EIF2AK2 ; GRK6 ; MAPK1; Metabolism PLK1; AKT2 ; CDK8; MAPK8; MAPK3; PRKCD ; PRKAA1; PBEF1 ; MAPKO ; CDK2; PIM1; DYRK1A ; MAP2K2; MAP2K1; PAK3 ; NT5E ; TTK ; CSNK1A1; BRAF ; SGK Chemokine Signaling CXCR4 ; ROCK2 ; MAPK1; PTK2; FOS ; CFL1; GNAQ ; CAMK2A ; CXCL12 ; MAPK8; MAPK3; KRAS ; MAPK13 ; RHOA ; CCR3 ; SRC ; PPP1CC ; MAPK14 ; NOX1 ; RAF1 ; MAP2K2; MAP2K1; JUN ; CCL2; PRKCA IL - 2 Signaling ELK1; MAPK1 ; PTPN11; AKT2 ; PIK3CA ; SYK ; FOS ; STAT5B ; PIK3CB ; PIK3C3 ; MAPK8; MAPK3; KRAS; SOCS1 ; STAT5A ; PIK3C2A ; LCK ; RAF1 ; MAP2K2 ; JAK1; AKT1 ; PIK3R1; MAP2K1; JUN ; AKT3 Synaptic Long Term PRKCE ; IGF1 ; PRKCZ ; PRDX6 ; LYN ; MAPK1; GNAS; PRKCI; GNAQ ; PPP2R1A ; IGF1R ; PRKD1; MAPK3 ; KRAS; GRN ; PRKCD ; NOS3; NOS2A ; PPP2CA ; YWHAZ ; RAF1; MAP2K2; PPP2R5C ; MAP2K1; PRKCA Estrogen Receptor TAF4B ; EP300 ; CARM1; PCAF ; MAPK1; NCOR2 ; Signaling SMARCA4 ; MAPK3; NRIP1 ; KRAS ; SRC ; NR3C1; HDAC3 ; PPARGC1A ; RBM9; NCOA3 ; RAF1; CREBBP ; MAP2K2; NCOA2; MAP2K1; PRKDC ; ESR1; ESR2 Protein Ubiquitination TRAF6 ; SMURF1 ; BIRC4 ; BRCA1; UCHL1; NEDD4 ; Pathway CBL ; UBE21; BTRC ; HSPA5; USP7 ; USP10 ; FBXW7; USP9X ; STUB1; USP22 ; B2M ; BIRC2; PARK2 ; USP8 ; USP1; VHL; HSP90AA1; BIRC3 IL -10 Signaling TRAF6 ; CCR1; ELK1 ; IKBKB ; SP1 ; FOS ; NFKB2; MAP3K14 ; MAPK8 ; MAPK13 ; RELA ; MAPK14 ; TNF ; IKBKG ; RELB ; MAP3K7; JAK1; CHUK ; STAT3; NFKB1 ; JUN ; IL1R1; IL6 VDR /RXR Activation PRKCE ; EP300 ; PRKCZ ; RXRA ; GADD45A ; HES1; NCOR2; SP1 ; PRKCI; CDKN1B ; PRKD1; PRKCD ; RUNX2; ; YY1; NCOA3 ; CDKN1A ; NCOA2; SPP1; LRP5 ; CEBPB ; FOXO1; PRKCA TGF- beta Signaling EP300 ; SMAD2; SMURF1 ; MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS ; MAPK9 ; RUNX2; SERPINE1 ; RAF1 ; MAP3K7 ; CREBBP ; MAP2K2; MAP2K1; TGFBR1; SMAD4 ; JUN ; SMAD5 Toll- like Receptor Signaling IRAK1; EIF2AK2; MYD88 ; TRAF6 ; PPARA ; ELK1; IKBKB ; FOS ; NFKB2; MAP3K14 ; MAPK8; MAPK13 ; RELA ; TLR4 ; MAPK14 ; IKBKG ; RELB ; MAP3K7 ; CHUK ; NFKB1 ; TLR2 ; JUN p38 MAPK Signaling HSPB1 ; IRAK1 ; TRAF6 ; MAPKAPK2; ELK1 ; FADD ; FAS ; CREB1; DDIT3 ; RPS6KA4 ; DAXX ; MAPK13 ; TRAF2 ; MAPK14 ; TNF ; MAP3K7 ; TGFBR1; MYC ; ATF4 ; IL1R1; SRF ; STAT1 / TRK Signaling NTRK2; MAPK1; PTPN11 ; PIK3CA ; CREB1; FOS; PIK3CB ; PIK3C3 ; MAPK8; MAPK3; KRAS ; PIK3C2A ; RAF1; MAP2K2 ; AKT1 ; PIK3R1; PDPK1; MAP2K1; CDC42 ; JUN ; ATF4 FXR /RXR Activation INS ; PPARA ; FASN ; RXRA ; AKT2 ; SDC1; MAPK8; APOB ; MAPK10 ; PPARG ; MTTP ; MAPK9 ; PPARGC1A ; TNF; CREBBP ; AKT1; SREBF1; FGFR4 ; AKT3 ; FOXO1 Synaptic Long Term PRKCE ; RAP1A ; EP300 ; PRKCZ ; MAPK1; CREB1; Potentiation PRKCI; GNAQ ; CAMK2A ; PRKD1; MAPK3 ; KRAS ; PRKCD ; PPP1CC ; RAF1; CREBBP ; MAP2K2 ; MAP2K1; ATF4 ; PRKCA RAP1A ; EP300 ; HDAC4 ; MAPK1; HDAC5 ; CREB1; CAMK2A ; MYH9; MAPK3 ; HDAC2; HDAC7A ; HDAC11 ; HDAC9 ; HDAC3; CREBBP ; CALR ; CAMKK2; ATF4 ; HDAC6 US 9 , 856 , 497 B2 95 96 TABLE 5 -continued CELLULAR FUNCTION GENES EGF Signaling ELK1; MAPK1; EGFR ; PIK3CA ; FOS ; PIK3CB ; PIK3C3; MAPK8; MAPK3 ; PIK3C2A ; RAF1; JAK1; PIK3R1; STAT3 ; MAP2K1; JUN ; PRKCA ; SRF ; STAT1 Hypoxia Signaling in the EDN1; PTEN ; EP300 ; NQO1; UBE21; CREB1 ; ARNT ; Cardiovascular System HIF1A ; SLC2A4 ; NOS3 ; TP53 ; LDHA ; AKT1; ATM ; VEGFA ; JUN ; ATF4 ; VHL ; HSP90AA1 LPS/ IL - 1 Mediated Inhibition IRAK1; MYD88 ; TRAF6 ; PPARA ; RXRA ; ABCA1; of RXR Function MAPK8; ALDH1A1; GSTP1 ; MAPK9 ; ABCB1; TRAF2 ; TLR4 ; TNF ; MAP3K7; NR1H2 ; SREBF1; JUN ; IL1R1 LXR /RXR Activation FASN ; RXRA ; NCOR2; ABCA1; NFKB2; IRF3 ; RELA ; NOS2A ; TLR4 ; TNF ; RELB ; LDLR ; NR1H2; NFKB1; SREBF1 ; IL1R1; CCL2 ; IL6 ; MMP9 Amyloid Processing PRKCE ; CSNK1E ; MAPK1; CAPNS1 ; AKT2 ; CAPN2; CAPN1; MAPK3; MAPK13; MAPT ; MAPK14 ; AKT1; PSEN1 ; CSNK1A1 , GSK3B ; AKT3 ; APP IL - 4 Signaling AKT2 ; PIK3CA ; PIK3CB ; PIK3C3 ; IRS1; KRAS; SOCS1; PTPN6 ; NR3C1; PIK3C2A ; JAK1; AKT1 ; JAK2; PIK3R1; FRAP1 ; AKT3 ; RPS6KB1 Cell Cycle : G2/ M DNA EP300 ; PCAF ; BRCA1 ; GADD45A ; PLK1; BTRC ; Damage Checkpoint CHEK1; ATR ; CHEK2; YWHAZ ; TP53 ; CDKN1A ; Regulation PRKDC ; ATM ; SFN ; CDKN2A Nitric Oxide Signaling in the KDR ; FLT1 ; PGF; AKT2 ; PIK3CA ; PIK3CB ; PIK3C3 ; Cardiovascular System CAV1; PRKCD ; NOS3; PIK3C2A ; AKT1; PIK3R1; VEGFA ; AKT3 ; HSP90AA1 Purine Metabolism NME2 ; SMARCA4 ; MYH9 ; RRM2; ADAR ; EIF2AK4 ; PKM2; ENTPD1 ; RAD51 ; RRM2B ; TJP2; RAD51C ; NT5E ; POLD1; NME1 CAMP- mediated Signaling RAP1A ; MAPK1; GNAS ; CREB1; CAMK2A ; MAPK3 ; SRC ; RAF1; MAP2K2; STAT3 ; MAP2K1; BRAF ; ATF4 Mitochondrial Dysfunction SOD2 ; MAPK8 ; CASP8 ; MAPK10 ; MAPK9 ; CASP9 ; PARK7; PSEN1; PARK2 ; APP ; CASP3 Notch Signaling HES1; JAG1; NUMB ; NOTCH4 ; ADAM17 ; NOTCH2 ; PSEN1; NOTCH3 ; NOTCH1; DLL4 Endoplasmic Reticulum Stress HSPA5 ; MAPK8; XBP1; TRAF2; ATF6 ; CASP9 ; ATF4 ; Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2; AICDA ; RRM2; EIF2AK4 ; ENTPD1; RRM2B ; NT5E ; POLD1; NME1 Parkinson 's Signaling UCHL1 ; MAPK8 ; MAPK13 ; MAPK14 ; CASP9 ; PARK7; PARK2; CASP3 Cardiac & Beta GNAS; GNAQ ; PPP2R1A ; GNB2L1; PPP2CA ; PPP1CC ; Signaling PPP2R5C Glycolysis/ Gluconeogenesis HK2; GCK ; GPI; ALDH1A1; PKM2 ; LDHA ; HK1 Interferon Signaling IRF1 ; SOCS1; JAK1; JAK2 ; IFITM1; STAT1 ; IFIT3 Sonic Hedgehog Signaling ARRB2 ; SMO ; GLI2 ; DYRK1A ; GLI1 ; GSK3B ; DYRKIB Glycerophospholipid PLD1 ; GRN ; GPAM ; YWHAZ ; SPHK1; SPHK2 Metabolism Phospholipid Degradation PRDX6 ; PLD1; GRN ; YWHAZ ; SPHK1; SPHK2 Tryptophan Metabolism SIAH2; PRMT5 ; NEDD4 ; ALDH1A1; CYP1B1; SIAH1 Degradation SUV39H1; EHMT2 ; NSD1; SETD7; PPP2R5C Nucleotide Excision Repair ERCC5; ERCC4; XPA : XPC : ERCC1 Pathway Starch and Sucrose UCHL1 ; HK2 ; GCK ; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2; GCK ; HK1 Metabolism PRDX6 ; GRN ; YWHAZ ; CYP1B1 Signaling CSNK1E ; CREB1; ATF4 ; NR1D1 Coagulation System BDKRB1; F2R ; SERPINE1; F3 Dopamine Receptor Signaling PPP2R1A ; PPP2CA ; PPP1CC ; PPP2R5C Glutathione Metabolism IDH2 ; GSTP1; ANPEP ; IDH1 Glycerolipid Metabolism ALDH1A1; GPAM ; SPHK1; SPHK2 Metabolism PRDX6 ; GRN ; YWHAZ ; CYP1B1 Methionine Metabolism DNMT1 ; DNMT3B ; AHCY ; DNMT3A Pyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and Proline ALDH1A1; NOS3 ; NOS2A Metabolism Signaling PRDX6 ; GRN ; YWHAZ Fructose and Mannose HK2; GCK ; HK1 Metabolism Galactose Metabolism HK2 ; GCK , HK1 Stilbene, Coumarine and PRDX6 ; PRDX1; TYR Lignin Biosynthesis Antigen Presentation Pathway CALR ; B2M Biosynthesis of Steroids NQO1; DHCR7 Butanoate Metabolism ALDH1A1 ; NLGN1 Citrate Cycle IDH2; IDH1 Fatty Acid Metabolism ALDH1A1 ; CYP1B1 Glycerophospholipid PRDX6 ; CHKA Metabolism